U.S. patent number 7,208,298 [Application Number 11/439,162] was granted by the patent office on 2007-04-24 for process for producing isoprenoid compounds by microorganisms and a method for screening compounds with antibiotic or weeding activity.
This patent grant is currently assigned to Kyowa Hakko Kogyo Co., Ltd.. Invention is credited to Shinichi Hashimoto, Tomohisa Kuzuyama, Koichiro Miyake, Hiroaki Motoyama, Akio Ozaki, Haruo Seto, Shunji Takahashi.
United States Patent |
7,208,298 |
Miyake , et al. |
April 24, 2007 |
Process for producing isoprenoid compounds by microorganisms and a
method for screening compounds with antibiotic or weeding
activity
Abstract
The present invention provides a process for producing
isoprenoid compounds or proteins encoded by DNA using DNA that
contains one or more of the DNA encoding proteins having activity
to improve efficiency in the biosynthesis of isoprenoid compounds
effective in pharmaceuticals for cardiac diseases, osteoporosis,
homeostasis, prevention of cancer, and immunopotentiation, health
food and anti-fouling paint products against barnacles; the DNA;
the protein; and a method for screening a substance with antibiotic
and weeding activities comprising screening a substance inhibiting
enzymatic reaction on the non-mevalonate pathway.
Inventors: |
Miyake; Koichiro (Yamaguchi,
JP), Hashimoto; Shinichi (Kanagawa, JP),
Motoyama; Hiroaki (Kanagawa, JP), Ozaki; Akio
(Tokyo, JP), Seto; Haruo (Tokyo, JP),
Kuzuyama; Tomohisa (Tokyo, JP), Takahashi; Shunji
(Chiba, JP) |
Assignee: |
Kyowa Hakko Kogyo Co., Ltd.
(Tokyo, JP)
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Family
ID: |
27288856 |
Appl.
No.: |
11/439,162 |
Filed: |
May 24, 2006 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20070004000 A1 |
Jan 4, 2007 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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10938613 |
Sep 13, 2004 |
7132268 |
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09673198 |
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6806076 |
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PCT/JP99/01987 |
Apr 14, 1999 |
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Foreign Application Priority Data
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Apr 14, 1998 [JP] |
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10-103101 |
Aug 5, 1998 [JP] |
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10-221910 |
Feb 15, 1999 [JP] |
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11-035739 |
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Current U.S.
Class: |
435/166; 435/189;
435/320.1; 536/23.2; 435/252.3; 435/183; 435/167 |
Current CPC
Class: |
C12N
9/1085 (20130101); C12N 15/52 (20130101); C12P
23/00 (20130101); C12Q 1/18 (20130101); C12P
7/66 (20130101); C12N 9/0006 (20130101); C12Y
202/01007 (20130101); C12N 9/1022 (20130101); G01N
2430/20 (20130101) |
Current International
Class: |
C12P
5/00 (20060101); C12N 9/02 (20060101) |
Field of
Search: |
;435/167,189,252.3,320.1,166 ;536/23.2 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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752714 |
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Jan 2000 |
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AU |
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757440 |
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Feb 2000 |
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AU |
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298 00 547 |
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Apr 1999 |
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DE |
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WO 97/43437 |
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Nov 1997 |
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WO |
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WO 00/00816 |
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Jan 2000 |
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WO |
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WO 00/08169 |
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Feb 2000 |
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WO |
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WO 00/17233 |
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Mar 2000 |
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WO |
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WO 00/34448 |
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Jun 2000 |
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WO |
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WO 00/42205 |
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Jul 2000 |
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WO |
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WO 00/44912 |
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Aug 2000 |
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WO |
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Primary Examiner: Saidha; Tekchand
Attorney, Agent or Firm: Fitzpatrick, Cella, Harper &
Scinto
Parent Case Text
This application is a divisional of application Ser. No. 10/938,613
filed Sep. 13, 2004, which in turn is a divisional of application
Ser. No. 09/673,198 filed Oct. 12, 2000 (now U.S. Pat. No.
6,806,076), which in turn is a National Phase of PCT Application
No. PCT/JP99/01987 filed Apr. 14, 1999.
Claims
The invention claimed is:
1. A process for producing an isoprenoid compound comprising the
steps of: introducing a vector containing DNA encoding a protein
comprising an amino acid sequence of SEQ ID NO:26 into a
prokaryotic host cell to produce a transformant; culturing the
transformant in a medium; allowing the transformant to produce and
accumulate the isoprenoid compound; and recovering the isoprenoid
compound.
2. A process for producing an isoprenoid compound comprising the
steps of: culturing a prokaryotic transformant harboring a vector
containing DNA encoding a protein comprising an amino acid sequence
of SEQ ID NO:26 in a medium; allowing the transformant to produce
and accumulate the isoprenoid compound; and recovering the
isoprenoid compound.
3. The process according to claim 1 or 2, wherein the DNA comprises
a nucleotide sequence of SEQ ID NO:27.
4. The process according to claim 1 or 2, wherein the isoprenoid
compound is selected from the group consisting of ubiquinone,
vitamin K.sub.2 and carotenoid.
5. The process according to claim 3, wherein the isoprenoid
compound is selected from the group consisting of ubiquinone,
vitamin K.sub.2 and carotenoid.
6. A process for producing an isoprenoid compound comprising the
steps of: introducing a vector containing DNA which hybridizes with
a nucleotide sequence consisting of SEQ ID NO:27 in the presence of
0.7 to 1.0 mol/l NaCl at 65.degree. C. followed by washing in a 0.1
to 2-fold SSC solution at 65.degree. C. and encodes a protein
having activity to catalyze a reaction to produce
2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose
5-phosphate into a prokaryotic host cell to produce a transformant;
culturing the transformant in a medium; allowing the transformant
to produce and accumulate the isoprenoid compound; and recovering
the isoprenoid compound.
7. A process for producing an isoprenoid compound comprising the
steps of: culturing a prokaryotic transformant harboring a vector
containing DNA that hybridizes with a nucleotide sequence
consisting of SEQ ID NO:27 in the presence of 0.7 to 1.0 mol/l NaCl
at 65.degree. C. followed by washing in a 0.1 to 2-fold SSC
solution at 65.degree. C. and encodes a protein having activity to
catalyze a reaction to produce 2-C-methyl-D-erythritol 4-phosphate
from 1-deoxy-D-xylulose 5-phosphate in a medium; allowing the
transformant to produce and accumulate the isoprenoid compound; and
recovering the isoprenoid compound.
8. The process according to claim 6 or 7, wherein the isoprenoid
compound is selected from the group consisting of ubiquinone,
vitamin K.sub.2 and carotenoid.
Description
TECHNICAL FIELD
The present invention relates to a method for producing isoprenoid
compounds using a transformant derived from a prokaryote; and a
method for screening substances having antibiotic or weeding
activity involved in a non-mevalonate pathway.
BACKGROUND ART
Isoprenoid is a general term for compounds having isoprene unit
consisting of 5 carbon atoms as a backbone structure. Isoprenoid is
biosynthesized by polymerization of isopentenyl pyrophosphate
(IPP). Various kinds of isoprenoid compounds are present in nature
and many of them are useful for humans.
For example, ubiquinone plays an important role in vivo as an
essential component of the electron transport system. The demand
for ubiquinone is increasing not only as a pharmaceutical effective
against cardiac diseases, but also as a health food in Western
countries.
Vitamin K, an important vitamin involved in the blood coagulation
system, is utilized as a hemostatic agent. Recently it has been
suggested that vitamin K is involved in osteo-metabolism, and is
expected to be applied to the treatment of osteoporosis.
Phylloquinone and menaquinone have been approved as
pharmaceuticals.
In addition, ubiquinone and vitamin K are effective in inhibiting
barnacles from clinging to objects, and so would make an excellent
additive to paint products to prevent barnacles from clinging.
Further, compounds called carotenoids having an isoprene backbone
consisting of 40 carbon atoms have antioxidant effect. Carotenoids
such as .beta.-carotene, astaxanthin, and cryptoxanthin are
expected to possess cancer preventing and immunopotentiating
activity.
As described above, isoprenoid compounds include many effective
substances. Establishment of an economical process for producing
these substances will be a huge benefit to the medical world and
society.
The process for producing isoprenoid compounds through fermentation
has already been examined, and examination of culture conditions,
strain breeding by mutagenesis, and improvement of yield by genetic
engineering techniques have been tested. However, the practical
results are limited to individual types of compounds, and there is
no known method effective for the isoprenoid compounds in
general.
Isopentenyl pyrophosphate (IPP), a backbone unit of isoprenoid
compounds, has been proved to be biosynthesized from acetyl-CoA via
mevalonic acid (mevalonate pathway) in eukaryotes, such as an
animal and yeast.
3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase is considered to
be a rate-limiting enzyme in the mevalonate pathway [Mol. Biol.
Cell, 5, 655 (1994)]. A test in yeast to improve the yield of
carotenoids by overexpression of HMG-CoA reductase has been
conducted [Misawa, et al., Summaries of Lectures on Carotenoids,
1997].
There is no knowledge which proves the presence of the mevalonate
pathway in prokaryotes. In many prokaryotes, another pathway, the
non-mevalonate pathway, has been found in which IPP is
biosynthesized via 1-deoxy-D-xylulose 5-phosphate produced by
condensation of pyruvic acid and glyceraldehyde 3-phosphate
[Biochem. J., 295, 517 (1993)]. It is suggested that
1-deoxy-D-xylulose 5-phosphate is converted to IPP via
2-C-methyl-D-erythritol 4-phosphate in an experiment using
.sup.13C-labelled substrate [Tetrahedron Lett. 38, 4769
(1997)].
In Escherichia coli, a gene encoding an enzyme, 1-deoxy-D-xylulose
5-phosphate synthase (DXS) which allows biosynthesis of
1-deoxy-D-xylulose 5-phosphate by condensation of pyruvic acid and
glyceraldehyde 3-phosphate, is identified [Proc. Natl. Acad. Sci.
USA, 94, 12857 (1997)]. Said gene is contained in an operon
consisting of four ORFs that include ispA encoding farnesyl
pyrophosphate synthase.
Further in Escherichia coli, the presence of the activity to
convert 1-deoxy-D-xylulose 5-phosphate to 2-C-methyl-D-erythritol
4-phosphate is known [Tetrahedron Lett. 39, 4509 (1998)].
At present there are no known description nor suggestion to improve
yield of an isoprenoid compound by genetically engineering these
genes contained in the operon.
Although knowledge about the non-mevalonate pathway in prokaryotes
has gradually increased, most enzymes involved therein and genes
encoding these enzymes still remain unknown.
In photosynthetic bacteria, there is a known process for
effectively producing ubiquinone-10 by introducing a gene for an
enzyme ubiC (uviC gene), which converts chorismate into
4-hydroxybenzoate, and a gene for p-hydroxybenzoate transferase
(ubiA) (Japanese Unexamined Patent Application 107789/96). However,
there is no example which improved the productivity of isoprenoid
compounds by genetically engineering genes for enzymes involved in
the non-mevalonate pathway.
Moreover, there is no knowledge about how prokaryotes will be
influenced when the reaction on the non-mevalonate pathway is
inhibited by mutagenesis or treating with drugs.
DISCLOSURE OF THE INVENTION
The object of this invention is to provide a process for producing
isoprenoid compounds comprising integrating DNA into a vector
wherein the DNA contains one or more DNA involved in biosynthesis
of isoprenoid compounds useful in pharmaceuticals for cardiac
diseases, osteoporosis, homeostasis, prevention of cancer, and
immunopotentiation, health food and anti-fouling paint products
against barnacles, introducing the resultant recombinant DNA into a
host cell derived from prokaryotes, culturing the obtained
transformant in a medium, allowing the transformant to produce and
accumulate isoprenoid compounds in the culture, and recovering the
isoprenoid compounds from said culture; a process for producing
proteins comprising integrating DNA into a vector wherein the DNA
contains one or more DNA encoding a protein having activity to
improve efficiency in the biosynthesis of isoprenoid compounds,
introducing the resultant recombinant DNA into a host cell,
culturing the obtained transformant in a medium, allowing the
transformant to produce and accumulate said protein in the culture,
and recovering said protein from the culture; the protein; and DNA
encoding the protein. A further object of this invention is to
provide a method of screening a substance having antibiotic and/or
weeding activities, which comprises screening the substance
inhibiting enzymatic reaction on the non-mevalonic acid
pathway.
The inventors have completed the invention by finding that the
productivity of isoprenoid can be improved by screening DNA capable
of improving the productivity for isoprenoid in prokaryotes, and
introducing the obtained DNA into prokaryotes.
That is, the first invention of the present application is a
process for producing isoprenoid compounds comprising integrating
DNA into a vector wherein the DNA contains one or more DNA selected
from the following (a), (b), (c), (d), (e) and (f): (a) a DNA
encoding a protein having activity to catalyze a reaction to
produce 1-deoxy-D-xylulose 5-phosphate from pyruvic acid and
glyceraldehyde 3-phosphate, (b) a DNA encoding farnesyl
pyrophosphate synthase, (c) a DNA encoding a protein that has an
amino acid sequence of SEQ ID NO:3, or a protein that has an amino
acid sequence wherein one to several amino acid residues are
deleted, substituted or added in the amino acid sequence of SEQ ID
NO:3 and has activity to improve efficiency in the biosynthesis of
isoprenoid compounds, (d) a DNA encoding a protein that has an
amino acid sequence of SEQ ID NO:4, or a protein that has an amino
acid sequence wherein one to several amino acid residues are
deleted, substituted or added in the amino acid sequence of SEQ ID
NO:4 and has activity to improve efficiency in the biosynthesis of
isoprenoid compounds, (e) a DNA encoding a protein having activity
to catalyze a reaction to produce 2-C-methyl-D-erythritol
4-phosphate from 1-deoxy-D-xylulose 5-phosphate, and (f) a DNA
encoding a protein that can hybridize under stringent conditions
with DNA selected from (a), (b), (c), (d) and (e), and has activity
substantially identical with that of the protein encoded by the
selected DNA; introducing the resultant recombinant DNA into a host
cell derived from prokaryotes, culturing the obtained transformant
in a medium; allowing the transformant to produce and accumulate
isoprenoid compounds in the culture; and recovering the isoprenoid
compounds from the culture.
Deletions, substitutions or additions of amino acid residues in
this specification can be carried out by site-directed mutagenesis,
which is a technique well-known prior to the filing of this
application. Further, the phrase "one to several amino acid
residues" means the number of amino acid residues, which can be
deleted, substituted, or added by site-directed mutagenesis, for
example, 1 to 5 amino acid residues.
The protein consisting of an amino acid sequence, which has
deletion, substitution or addition of one to several amino acid
residues, can be prepared according to the methods described in
Molecular Cloning: A Laboratory Manual, Second Edition, ed.
Sambrook, Fritsch, and Maniatis, Cold Spring Harbor Laboratory
Press, 1989 (hereinafter referred to as Molecular Cloning, Second
Edition), Current Protocols in Molecular Biology, John Wiley &
Sons (1987 1997), Nucleic Acids Research, 10, 6487 (1982), Proc.
Natl. Acad. Sci., USA, 79, 6409 (1982), Gene, 34, 315 (1985),
Nucleic Acids Research, 13, 4431 (1985), and Proc. Natl. Acad. Sci
USA, 82, 488 (1985), etc.
The above-mentioned DNA encoding a protein, which catalyzes a
reaction to produce 1-deoxy-D-xylulose 5-phosphate from pyruvic
acid and glyceraldehyde 3-phosphate, is for example, a DNA encoding
a protein, which has an amino acid sequence of SEQ ID NO:1, 26 or
28, or a DNA encoding a protein which has an amino acid sequence
wherein one to several amino acid residues are deleted, substituted
or added in the amino acid sequence of SEQ ID NO:1, 26, or 28 and
has activity to catalyze a reaction to produce 1-deoxy-D-xylulose
5-phosphate from pyruvic acid and glyceraldehyde 3-phosphate.
Examples of such a DNA include a DNA having an nucleotide sequence
of SEQ ID NO:6 or a DNA having a nucleotide sequence of SEQ ID
NO:27 or 29.
Examples of a DNA encoding farnesyl pyrophosphate synthase include
a DNA encoding a protein having an amino acid sequence of SEQ ID
NO:2 or a DNA encoding a protein, which has an amino acid sequence
wherein one to several amino acid residues are deleted, substituted
or added in the amino acid sequence of SEQ ID NO:2 and has
enzymatic activity to produce farnesyl pyrophosphate. A specific
example is a DNA having a nucleotide sequence of SEQ ID NO:7.
A specific example of the DNA encoding a protein having an amino
acid sequence of SEQ ID NO:3 is a DNA having a nucleotide sequence
of SEQ ID NO:8.
Further a specific example of the DNA encoding a protein having an
amino acid sequence of SEQ ID NO:4 is a DNA having a nucleotide
sequence of SEQ ID NO:9.
Examples of the DNA encoding a protein having activity to catalyze
a reaction to produce 2-C-methyl-D-erythritol 4-phosphate from
1-deoxy-D-xylulose 5-phosphate include a DNA encoding a protein,
which has an amino acid sequence of SEQ ID NO:5 or 30, or a DNA
encoding a protein, which has an amino acid sequence wherein one to
several amino acid residues are deleted, substituted or added in
the amino acid sequence of SEQ ID NO:5 or 30 and has activity to
catalyze the reaction to produce 2-C-methyl-D-erythritol
4-phosphate from 1-deoxy-D-xylulose 5-phoshphate.
Specifically, such a DNA is one having a nucleotide sequence of SEQ
ID NO:10 or 31.
The above phrase "DNA . . . that can hybridize under stringent
conditions" means a DNA that can be obtained by colony
hybridization, plaque hybridization, Southern Blotting or the like
using the above DNA or fragments of the DNA as a probe. Such a DNA
can be identified by performing hybridization using a filter with
colony--or plaque-derived DNA, or fragments of the DNA immobilized
thereon, in the presence of 0.7 to 1.0 mol/l NaCl at 65.degree. C.,
followed by washing the filter using about 0.1 to 2-fold SSC
solution (the composition of SSC solution at 1-fold concentration
is consisted of 150 mol/l sodium chloride, 15 mol/l sodium citrate)
at 65.degree. C.
Hybridization can be carried out according to the methods described
in Molecular Cloning, Second Edition. Examples of DNA capable of
hybridizing include a DNA that shares at least 70% or more
homology, preferably, 90% or more homology with a nucleotide
sequence selected from SEQ ID NOS:1, 2, 3, 4, and 5.
Examples of isoprenoid compounds include ubiquinone, vitamin
K.sub.2, and carotenoids.
The second invention of this application is a protein having
activity to improve efficiency in the biosynthesis of isoprenoid
compounds and selected from the following (a), (b) and (c): (a) a
protein having an amino acid sequence of SEQ ID NO:3, or a protein
having an amino acid sequence wherein one to several amino acid
residues are deleted, substituted or added in the amino acid
sequence of SEQ ID NO:3 (b) a protein having an amino acid sequence
of SEQ ID NO:4, or a protein having an amino acid sequence wherein
one to several amino acid residues are deleted, substituted or
added in the amino acid sequence of SEQ ID NO:4, and (c) a protein
having an amino acid sequence of SEQ ID NO:5, or a protein having
an amino acid sequence wherein one to several amino acid residues
are deleted, substituted or added in the amino acid sequence of SEQ
ID NO:5.
The third invention of this application is a process for producing
a protein having activity to improve efficiency in the biosynthesis
of isoprenoid compounds comprising integrating DNA encoding the
protein described in the second invention above into a vector,
introducing the resultant recombinant DNA into a host cell,
culturing the obtained transformant in a medium, allowing the
transformant to produce and accumulate the protein in the culture,
and recovering the protein from the culture.
The transformants above include microorganisms belonging to the
genus Escherichia, Rhodobacter or Erwinia.
The fourth invention of this application is a DNA encoding a
protein having activity to improve efficiency in the biosynthesis
of isoprenoid compounds selected from the following (a), (b), (c),
(d), (e), (f) and (g): (a) a DNA encoding a protein having an amino
acid sequence of SEQ ID NO:3, (b) a DNA encoding a protein having
an amino acid sequence of SEQ ID NO:4, (c) a DNA encoding a protein
having an amino acid sequence of SEQ ID NO:5, (d) a DNA having a
nucleotide sequence of SEQ ID NO:8, (e) a DNA having a nucleotide
sequence of SEQ ID NO:9, (f) a DNA having a nucleotide sequence of
SEQ ID NO:10, and (g) a DNA that can hybridize with any one of DNA
described in (a) to (f) under stringent conditions.
The fifth invention of this application is a method for screening a
substance having antibiotic activity comprising screening a
substance that inhibits the reaction of a protein having activity
of an enzyme selected from those present on the non-mevalonate
pathway in which 1-deoxy-D-xylulose 5-phosphate biosynthesized from
pyruvic acid and glyceraldehyde 3-phosphate is converted to
2-C-methyl-D-erythritol 4-phosphate from which isopentenyl
pyrophosphate is biosynthesized.
The sixth invention of this application is a method for screening a
substance having weeding activity comprising screening a substance
that inhibits the reaction of a protein having activity of an
enzyme selected from those present on the non-mevalonate pathway in
which 1-deoxy-D-xylulose 5-phosphate biosynthesized from pyruvic
acid and glyceraldehyde 3-phosphate is converted to
2-C-methyl-D-erythritol 4-phosphate from which isopentenyl
pyrophosphate is biosynthesized.
Examples of the proteins in the fifth and sixth inventions above
include a protein of the following (a) or (b): (a) a protein having
activity to catalyze a reaction to produce 1-deoxy-D-xylulose
5-phosphate from pyruvic acid and glyceraldehyde 3-phosphate, or
(b) a protein having activity to catalyze a reaction to produce
2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose
5-phosphate.
Examples of the proteins catalyzing the reaction to produce
1-deoxy-D-xylulose 5-phosphate from pyruvic acid and glyceraldehyde
3-phosphate include a protein having an amino acid sequence of SEQ
ID NO:1, or a protein having an amino acid sequence wherein one to
several amino acid residues are deleted, substituted or added in
the amino acid sequence of SEQ ID NO:1, and having activity to
catalyze 1-deoxy-D-xylulose 5-phosphate from pyruvic acid and
glyceraldehyde 3-phosphate.
Examples of the proteins having activity to catalyze the reaction
to produce 2-C-methyl-D-erythritol 4-phosphate from
1-deoxy-D-xylulose 5-phosphate include a protein having an amino
acid sequence of SEQ ID NO:5, or a protein having an amino acid
sequence wherein one to several amino acid residues are deleted,
substituted or added in the amino acid sequence of SEQ ID NO:5, and
having activity to catalyze the reaction to produce
2-C-methyl-D-erythritol A-phosphate from 1-deoxy-D-xylulose
5-phosphate.
The seventh invention of this invention is a substance, which has
antibiotic activity and is obtained by the screening method in the
fifth invention above. Known substances obtained by the above
screening method are not included in this invention.
The inventors have focused on structural similarity of fosmidomycin
[3-(N-formyl-N-hydroxyamino)propylphosphonic acid] to
2-C-methyl-D-erythritol 4-phosphate, a reaction product from
1-deoxy-D-xylulose 5-phosphate reductoisomerase reaction, or a
reaction intermediate assumed to be produced in this enzymatic
reaction.
Based on the assumption that fosmidomycin has activity to inhibit
1-deoxy-D-xylulose 5-phosphate reductoisomerase and antibiotic
activity, the inventors have conducted experiments on the screening
method of the fifth invention and also described in the following
Example 10. As a result, the inventors found that fosmidomycin is a
substance having the activity to inhibit 1-deoxy-D-xylulose
5-phosphate reductoisomerase and antibiotic activity, and in
addition, verified the adequacy of the screening method of the
fifth invention above. However, known compound fosmidomycin is
excluded from this invention.
The eighth invention of this invention is a substance, which has
weeding activity and obtained through the screening method of the
sixth invention above. As described above, any substance that is
obtained from the screening method and already known is excluded
from this invention.
Hereinafter a more detailed explanation of this invention will be
given.
I. Cloning of DNA Encoding a Protein Involved in Biosynthesis of
Isoprenoid Compounds
(1) Cloning of DNA Encoding a Protein Involved in Biosynthesis of
Isoprenoid Compounds Using a Nucleotide Sequence of DNA (DXS Gene)
Encoding DXS
Using information on previously-determined nucleotide sequences of
E. coli chromosome and DXS gene [Proc. Natl. Acad. Sci. USA., 94,
12857 (1997)], a DNA region containing DXS gene or genes
neighboring DXS gene is obtained by cloning with PCR method from E.
coli [Science, 230, 1350 (1985)].
An example of information on a nucleotide sequence containing DXS
gene is the nucleotide sequence of SEQ ID NO:11.
A concrete example of methods for cloning the DNA region containing
DXS gene is as follows.
Escherichia coli, such as an E. coli XL1-Blue strain (available
from TOYOBO CO., LTD.), is cultured in a suitable medium for
Escherichia coli, for example, LB liquid medium [containing 10 g of
Bactotrypton (manufactured by Difco Laboratories), 5 g of Yeast
extracts (manufactured by Difco Laboratories), 5 g of NaCl per
liter of water, and adjusted to pH 7.2] according to standard
techniques.
After culturing, cells were recovered from the culture by
centrifugation.
Chromosomal DNA is isolated from the obtained cells according to a
known method, described in, for example, Molecular Cloning, Second
Edition.
Using information on a nucleotide sequence of SEQ ID NO:11, a sense
primer and an antisense primer, which contain DXS gene or a
nucleotide sequence corresponding to the DNA region of genes
neighboring DXS gene, are synthesized with a DNA synthesizer.
To introduce the amplified DNA fragments into a plasmid after
amplification with PCR, it is preferable to add recognition sites
appropriate for restriction enzymes, e.g., BamHI, and EcoRI to the
5' ends of sense and antisense primers.
Examples of a combination of the sense and antisense primers
include a DNA having a combination of nucleotide sequences: SEQ ID
NOS:12 and 13, SEQ ID NOS:14 and 15, SEQ ID NOS:12 and 16, SEQ ID
NOS:17 and 18, SEQ ID NOS:19 and 13, or SEQ ID NOS:22 and 23.
Using the chromosomal DNA as a template, PCR is carried out with
DNA Thermal Cycler (manufactured by Perkin Elmer Instruments, Inc.
Japan) using the primers; TaKaRa LA-PCR.TM. Kit Ver. 2
(manufactured by TAKARA SHUZO CO., LTD.) or Expand.TM.
High-Fidelity PCR System (manufactured by Boehringer Manheim
K.K.)
In a reaction condition for PCR, PCR is carried out by 30 cycles,
in the case of amplifying a DNA fragment of 2 kb or less, one cycle
consisting of reaction at 94.degree. C. for 30 seconds, 55.degree.
C. for 30 seconds to 1 minute, and 72.degree. C. for 2 minutes; in
the case of amplifying a DNA fragment of more than 2 kb, one cycle
consisting of reaction at 98.degree. C. for 20 seconds, and
68.degree. C. for 3 minutes; then followed by the reaction at
72.degree. C. for 7 minutes.
The amplified DNA fragments are cut at sites the same as the
restriction enzyme sites added to the above primers, and are
fractionated and collected by using agarose gel electrophoresis,
sucrose density-gradient centrifugation and the like.
For cloning the amplified DNA obtained above, an appropriate
cloning vector is digested with restriction enzymes creating the
cohensive ends which are able to ligate with the amplified DNA
fragment. Using a recombinant DNA obtained by ligating the above
amplified DNA with the cloning vector, Escherichia coli, e.g., E
coil DH5 .alpha. (available from TOYOBO CO., LTD.) is
transformed.
As a cloning vector for cloning the amplified DNA, any cloning
vectors including phage vectors and plasmic vectors, which can
autonomously replicate in E.coli K12, can be used. Expression
vectors for E coli can be used as cloning vectors. Concrete
examples of the cloning vectors include ZAP Express [manufactured
by Stratagene, Strategies, 5, 58 (1992)], pBluescript II SK(+)
[Nucleic Acids Research, 17, 9494 (1989)], Lamdba ZAP II
(manufactured by Stratagene, .lamda.gt10, .lamda.gt11 (DNA Cloning,
A Practical Approach, 1, 49 (1985)), .lamda.TriplEx (manufactured
by Clonetec), .lamda.ExCell (manufactured by Pharmacia), pT7T318U
(manufactured by Pharmacia), pcD2 [H. Okayama and P. Berg; Mol.
Cell. Biol., 3, 280 (1983)], pMW218 (manufactured by WAKO PURE
CHEMICAL INDUSTRIES, LTD), pUC 118 (manufactured by TAKARA SHUZO
CO., LTD.), pEG400 [J Bac, 172, 2392 (1990)], and pQE-30
(manufactured by Qiagen, Inc.).
A plasmid DNA containing a DNA of interest can be obtained from the
resultant transformant according to standard techniques, such as
those describe in Molecular Cloning, Second Edition, Current
Protocols in Molecular Biology, Supplement 1 to 38, John Wiley
& Sons (1987 1997), DNA Cloning 1: Core Techniques, A Practical
Approach, Second Edition, Oxford University Press (1995).
A plasmid DNA containing a DNA encoding a protein having activity
to catalyze the reaction to produce 1-deoxy-D-xylulose 5-phosphate
from pyruvic acid and glyceraldehyde 3-phosphate, a DNA encoding
farnesyl pyrophosphate synthase, a DNA encoding a protein having an
amino acid sequence of SEQ ID NO:3, a DNA encoding a protein having
an amino acid sequence of SEQ ID NO:4 or the like; and a plasmid
DNA containing one or more DNAs above, can be obtained by the above
methods.
Such plasmids include plasmid pADO-1 that contains all of the DNA
above, plasmid pDXS-1 or pQEDXS-1 that contains a DNA having a
nucleotide sequence of SEQ ID NO:6, plasmid pISP-1 that contains a
DNA having a nucleotide sequence of SEQ ID NO:7, plasmid pXSE-1
that contains a DNA having a nucleotide sequence of SEQ ID NO:8,
and plasmid pTFE-1 that contains a DNA having a nucleotide sequence
of SEQ ID NO:9.
Using the nucleotide sequences of DNA fragments derived from E.
coli, which have been inserted into these plasmids, homologues of
the DNA can be obtained from other prokaryotes, such as
microorganisms belonging to the genus Rhodobacter, in the same
manner as described above.
(2) Cloning of DNA Encoding a Protein Having Activity to Complement
Methylerythritol-requiring Mutant of E. coli (Gene Complementing
Methylerythritol-requiring Mutant)
Construction of E. coli Methylerythritol-requiring Mutant
Escherichia coli, such as E. coli W3110 (ATCC14948), is cultured
according to standard techniques.
After culturing, cells are recovered from the obtained culture by
centrifugation.
The obtained cells are washed with an appropriate buffer agent,
such as 0.05 mol/l Tris-maleate buffer (pH 6.0). Then the cells are
suspended in the same buffer such that the cell density is 10.sup.4
to 10.sup.10 cells/ml.
Mutagenesis is carried out by standard techniques using the
suspension. In such a standard technique, for example, NTG is added
to the suspension to a final concentration of 600 mg/l, and then
the mixture is maintained for 20 minutes at room temperature.
This suspension after mutagenesis is spread on minimal agar medium
supplemented with 0.05 to 0.5% methylerythritol and cultured.
An example of minimal agar medium is M9 medium (Molecular Cloning,
Second Edition) supplemented with agar.
Methylerythritol that is chemically synthesized according to the
method described in Tetrahedron Letters, 38, 35, 6184 (1997) maybe
used.
Colonies grown after culturing are replicated on minimal agar media
and minimal agar media each containing 0.05 to 0.5%
methylerythritol. The mutant of interest, which requires
methylerythritol to grow, is selected. That is, a strain capable of
growing on minimal agar media containing methylerythritol but not
on minimal agar media lacking methylerythritol is selected.
Strain ME 7 is an example of the resultant
methylerythritol-requiring mutant obtained by the above
manipulations.
3 Cloning of the Gene Complementing Methylerythritol-requiring
Nature
Echerichia coli, such as E. coli W3110 (ATCC14948), is inoculated
into culture media, e.g., LB liquid medium, then cultured to the
logarithmic growth phase by standard techniques.
Cells are collected from the resultant culture by
centrifugation.
Chromosomal DNA is isolated and purified from the obtained cells
according to standard techniques, such as those described in
Molecular Cloning, Second Edition. The chromosomal DNA obtained by
the method described in (1) above can be used as isolated and
purified chromosomal DNA.
An appropriate amount of the chromosomal DNA is partially digested
with an appropriate restriction enzyme, such as Sau 3 A I. The
digested DNA fragments are fractionated by according to standard
techniques, such as sucrose density-gradient centrifugation (26,000
rpm, 20.degree. C., 20 hr).
The DNA fragments obtained by the above fractionation, 4 to 6 kb
each, are ligated to a vector, e.g., pMW118 (Nippon Gene), which
has been digested with an appropriate restriction enzyme to
construct a chromosomal DNA library.
The methylerythritol-requiring mutant isolated in above, such as
the strain ME 7, is transformed using the ligated DNA according to
standard techniques, e.g., those described in Molecular Cloning,
Second Edition.
The resulting transformants are spread on minimal agar media
supplemented with a drug corresponding to a drug-resistant gene
carried by the vector, such as M9 agar medium containing 100
.mu.g/l of ampicillin, then cultured overnight at 37.degree. C.
Thus, transformants that have recovered their methylerythritol
requirement can be selected by the method above.
Plasmids are extracted from the resultant transformants by standard
techniques. Examples of a plasmid that can allow the transformants
to recover their methylerythritol requirement are pMEW73 and
pQEDXR.
The nucleotide sequence of the DNA integrated into the plasmid is
sequenced.
An example of such a nucleotide sequence is a sequence containing a
nuclectide sequence for yaeM gene of SEQ ID NO:10. Using the
information on the nucleotide sequence for yaeM gene, homologues of
yaeM gene can be obtained from other prokaryotes or plants in the
same manner as described above.
II. Production of Proteins Having Activity to Improve Efficiency in
the Biosynthesis of Isoprenoid Compounds
To express the resulting DNA in a host cell, the DNA fragment of
interest is digested with restriction enzymes or deoxyribonucleases
into one with a proper length containing the gene. Next the
fragment is inserted into a downstream of a promoter region in an
expression vector. Then the expression vector is introduced into a
host cell appropriate for the expression vector.
Any host cell that can express the gene of interest can be used.
Examples of the host cell include bacteria belonging to the genera
Escherichia, Serratia, Corynebacterium, Brevibacterium,
Pseudomonas, Bacillus, Microbacterium and the like, yeasts
belonging to the genera Kluyveromyces, Saccharomyces,
Schizosaccharomyces, Trichosporon, Schwanniomyces, and the like,
animal cells, and insect cells.
Expression vectors used herein can autonomously replicate in the
host cell above or be integrated into a chromosomal DNA, and
contain a promoter at the position to which the DNA of interest as
described above can be transcribed.
When a bacterium is used as a host cell, a preferable expression
vector for expression of the DNA above can autonomously replicate
in the bacterium and is a recombinant vector comprising a promoter,
ribosome binding sequence, the DNA above and a transcription
termination sequence. The expression vector may contain a gene to
regulate a promoter.
Examples of the expression vector include pBTtp2, pBTac1, pBTac2
(all of them are available from Boehringer Manheim K.K.), pKK233-2
(Pharmacia), pSE280 (Invitrogen), pGEMEX-1 (Promega), pQE-8
(Qiagen. Inc), pQE-30 (Qiagen. Inc), pKYP10 (Japanese Patent Laid
Open Publication No, 58-110600), pKYP200 (Agricultural Biological
Chemistry, 48, 669, 1984), pLSA1 (Agric. Biol. Chem, 53, 277,
1989), pGEL1 (Proc. Natl. Acad. Sci. USA, 82, 4306, 1985),
pBluescriptII SK+, pBluescriptII SK (-) (Stratagene), pTrS30 (FERM
BP-5407), pTrS32 (FERM BP-5408), pGEX (Pharmacia), pET-3 (Novagen),
pTerm2 (U.S. Pat. Nos. 4,686,191, 4,939,094, 5,160,735), pSupex,
pUB110, pTP5, pC194, pUC18 (Gene, 33, 103, 1985), pUC19 (Gene, 33,
103, 1985), pSTV28 (TAKARA SHUZO CO., LTD.), pSTV29 (TAKARA SHUZO
CO., LTD.), pUC118 (TAKARA SHUZO CO., LTD.), pPA1 (Japanese Patent
Laid Open Publication No. 63-233798), pEG400 (J. Bacteriol., 172,
2392, 1990), and pQE-30 (Qiagen. Inc).
Any promoter that can function in a host cell may be used. Examples
of such a promoter include promoters derived from Escherichia coli
or phages, such as trp promoter (P trp), lac promoter (P lac),
P.sub.L promoter, P.sub.R promoter, P.sub.SE promoter, SP01
promoter, SP02 promoter, and penP promoter. Furthermore, P
trp.times.2 promoter that is formed by joining two P trp in series,
and tac promoter, letI promoter, and lacT7 promoter, those
artificially designed and modified, can be used.
Any ribosome binding sequence that can function in a host cell can
be used. A preferable plasmid has a distance between Shine-Dalgarno
sequence and a starting codon appropriately adjusted, of for
example 6 to 18 bases long.
A transcription termination sequence is not always required for
expression of the DNA of interest. Preferably, a transcription
termination sequence is arranged immediately followed by a
structural gene.
Examples of the host cell used herein include microorganisms
belonging to the genera Escherichia, Corynebacterium,
Brevibacterium, Bacillus, Microbacterium, Serratia, Pseudomonas,
Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Arthrobacter,
Azobacter, Chromatium, Erwinia, Methylobacterium, Phormidium,
Rhodobacter, Rhodopseudomonas, Rhodospirillum, Scenedesmun,
Streptomyces, Synnecoccus, and Zymomonas. Preferable host cells
include microorganisms belonging to the genera Escherichia,
Corynebacterium, Brevibacterium, Bacillus, Pseudomonas,
Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Arthrobacter,
Azobacter, Chromatium, Erwinia, Methylobacterium, Phormidium,
Rhodobacter, Rhodopseudomonas, Rhodospirillum, Scenedesmun,
Streptomyces, Synnecoccus and Zymomonas.
More specific examples of the host cell include Escherichia coli
XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH5.alpha.,
Escherichia coli DH5a, Escherichia coli MC1000, Escherichia coli
KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia
coli HB101, Escherichia coli No. 49, Escherichia coli W3110,
Escherichia coli NY49, Escherichia coli MP347, Escherichia coli
NM522, Bacillus subtilis, Bacillus amyloliquefacines,
Brevibacterium ammoniagenes, Brevibacterium immariophilum
ATCC14068, Brevibacterium saccharolyticum ATCC14066, Brevibacterium
flavum ATCC14067, Brevibacterium lactofermentum ATCC13869,
Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum
ATCC14297, Corynebacterium acetoacidophilum ATCC13870,
Microbacterium ammoniaphilum ATCC15354, Serratia ficaria, Serratia
fonticola, Serratia liquefaciens, Serratia marcescens, Pseudomonas
sp. D-0110, Agrobacterium radiobacter, Agrobacterium rhizogenes,
Agrobacterium rubi, Anabaena cylindrica, Anabaena doliolum, Anbaena
flos-aquae, Arthrobacter aurescens, Arthrobacter citreus,
Arthrobacter globformis, Arthrobacter hydrocarboglutamicus,
Arthrobacter mysorens, Arthrobacter nicotianae, Arthrobacter
paraffineus, Arthrobacter protophormiae, Arthrobacter
roseoparaffinus, Arthrobacter sulfureus, Arthrobacter ureafaciens,
Chromatium buderi, Chromatium tepidum, Chromatium vinosum,
Chromatium warmingii, Chromatium fluviatile, Erwinia uredovora,
Erwinia carotovora, Erwnia ananas, Erwinia herbicola, Erwinia
punctata, Erwinia terreus, Mehylobacterium rhodesianum,
Methylobacterium extorquens, Phomidium sp. ATCC29409, Rhodobacter
capsulatus, Rhodobacter sphaeroides, Rhodopseudomonas blastica,
Rhodopseudomonas marina, Rhodopseudomonas palustris, Rhodospirillum
rubrum, Rhodospirillum salexigens, Rhodospirillum salinarum,
Streptomyces ambofaciens, Streptomyces aureofaciens, Streptomyces
aureus, Streptomyces fungicidicus, Streptomyces griseochromogenes,
Streptomyces griseus, Streptomyces lividans, Streptomyces
olivogriseus, Streptomyces rameus, Streptomyces tanashiensis,
Streptomyces vinaceus, and Zymomonas mobilis.
Any method to introduce a recombinant vector into the host cell as
described above may be used. Examples of such a method include a
method using calcium ions (Proc. Natl. Acad. Sci. USA, 69, 2110,
1972), protoplast method (Japanese Patent Laid Open Publication No.
63-2483942), or methods described in Gene, 17, 107 (1982) or
Molecular & General Genetics, 168, 111 (1979).
When yeast is used as a host cell, expression vectors are, for
example, YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419),
pHS19, and pHS15.
Any promoter that can function in yeast can be used. Examples of
such a promoter include PH05 promoter, PGK promoter, GAP promoter,
ADH promoter, gal 1 promoter, gal 10 promoter, heat shock protein
promoter, MF .alpha.1 promoter, and CUP1 promoter.
Host cells used herein include Saccharomyces cerevisae,
Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon
pullulans, and Schwanniomyces alluvius.
Any method to introduce a recombinant vector, that is, to introduce
DNA into yeast may be used. Examples of such methods include
Electroporation (Methods. Enzymol., 194, 182, 1990), Spheroplast
method (Proc. Natl. Acad. Sci. USA, 75, 1929 (1978)), lithium
acetate method (J. Bacteriol., 153, 163 (1983)), and methods
described in Proc. Natl. Acad. Sci. USA, 75, 1929 (1978).
When an animal cell is used as a host cell, expression vectors are,
for example, pcDNAI, pcDM8 (Funakoshi Co., Ltd), pAGE107 [Japanese
Patent Laid Open Publication No. 3-22979; Cytotechnology, 3, 133
(1990)], pAS3-3 [Japanese Patent Laid Open Publication No.
2-227075, pCDM8 (Nature, 329, 840 (1987)), pcDNAI/Amp (Invitrogen),
pREP4 (Invitrogen), pAGE103 [J. Biochem. 101, 1307 (1987)], and
pAGE210.
Any promoter that can function in an animal cell may be used.
Examples of such promoters include a promoter for IE (immediate
early) gene of cytomegalovirus (human CMV), SV40 initial promoter,
retrovirus promoter, metallothionein promoter, heat shock promoter,
and SR .alpha. promoter. Moreover, an enhancer of human CMV IE gene
may be used together with a promoter.
Host cells used herein are, for example, Namalwa cells, HBT5637
(Japanese Patent Laid Open Publication No. 63-299), COS1 cells,
COS7 cells, and CHO cells.
Any method to introduce a recombinant vector into an animal cell,
that is, to introduce DNA into an animal cell may be used. Examples
of such methods include Electroporation [Cytotechnology, 3, 133
(1990)], calcium phosphate method (Japanese Patent Laid Open
Publication No. 2-227075), lipofection [Proc. Natl. Acad. Sci.,
USA, 84, 7413 (1987)], and methods described in Virology, 52, 456
(1973). Recovery and culture of the transformant can be carried out
according to methods described in Japanese Patent Laid Open
Publication No. 2-227075 and Japanese Patent Laid Open Publication
No. 2-257891.
When an insect cell is used as a host cell, proteins can be
expressed according to methods described in, such as Baculovirus
Expression Vectors, A Laboratory Manual, Current Protocols in
Molecular Biology Supplement 1 38 (1987 1997), and Bio/Technology,
6, 47 (1988).
That is, a vector for introducing a recombinant gene and
Baculovirus are co-transduced into an insect cell to obtain a
recombinant virus in the culture supernatant of the insect cell.
Then an insect cell is infected with the recombinant virus,
resulting in expression of the protein of interest.
Examples of the vectors to transfer genes include pVL1392, pVL1393,
pBlueBacIII (all of which are manufactured by Invitrogen).
Baculoviruses used herein are, for example, Autographa californica
nuclear polyhedrosis virus that infects Barathra insects.
Examples of the insect cells include ovarian cells of Spodoptera
frugiperda, Sf9, and Sf21 (Baculovirus Expression Vectors, A
Laboratory Manual (W. H. Freeman and Company, New York, 1992), and
of Trichoplusia ni, High 5 (Invitrogen).
Methods of co-transduction of the vector for transferring the
recombinant gene and the Baculovirus into an insect cell to prepare
a recombinant virus include calcium phosphate transfection
(Japanese Patent Laid Open Publication No. 2-227075) and,
lipofection [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].
Methods for expressing genes include secretory production, and
fusion protein expression according to the techniques shown in
Molecular Coning, Second Edition, in addition to direct
expression.
When the gene is expressed in yeasts, animal cells, or insect
cells, a protein to which sugar or a sugar chain is added, can be
obtained.
Proteins having activity to improve efficiency in the biosynthesis
of isoprenoid compounds can be produced by culturing a transformant
containing a recombinant DNA to which the above DNA has been
introduced in a medium, allowing the transformant to produce and
accumulate proteins having activity to improve efficiency in the
biosynthesis of isoprenoid compounds in the culture, then
collecting the proteins from the culture.
The transformants for producing proteins with activity to improve
efficiency in the biosynthesis of isoprenoid compounds of the
present invention, can be cultured by standard techniques to
culture a host cell.
When the transformant of this invention is prokaryote such as
Escherichia coli or eukaryote such as yeast, a medium for culturing
such transformants contains a carbon source, a nitrogen source, and
inorganic salts, which the microorganisms can assimilate, and
allows the transformant to grow efficiently. Ether natural media or
synthetic media can be used if they satisfy the above
conditions.
Any carbon source assimilable by the microorganisms may be used.
Such carbon sources include glucose, fructose, sucrose, and
molasses containing them, carbohydrates e.g., starch or
hydrolysates of starch, organic acids e.g., acetic acid and
propionic acid, and alcohols e.g., ethanol and propanol.
Examples of nitrogen sources include ammonia, salts of inorganic
acids or organic acids, e.g., ammonium chloride, ammonium sulfate,
ammonium acetate, and ammonium phosphate, other nitrogen-containing
compounds, peptone, meat extract, yeast extract, corn steep liquor,
casein hydrolysates, soybean meal and soybean meal hydrolysate,
various fermentation microorganic cells or their digests.
Examples of inorganic salts include potassium primary phosphate,
potassium secondary phosphate, magnesium phosphate, magnesium
sulfate, sodium chloride, ferrous sulfate, manganese sulfate,
copper sulfate, and calcium carbonate.
Culturing is carried out by shaking culture or submerged
aeration-agitation culture are carried out under aerobic
conditions. The preferable culture temperature ranges from 15 to
40.degree. C. The preferable culture period ranges from 16 hours to
7 days. The pH is kept within a range from 3.0 to 9.0 while
culturing. The pH is adjusted using inorganic or organic acid,
alkaline solutions, urea, calcium carbonate, ammonia or the
like.
If necessary, an antibiotics e.g., ampicillin or tetracycline may
be added to the media while culturing.
When microorganisms transformed with the expression vectors using
inducible promoters are cultured, inducers may be added to the
media if necessary. For example,
isopropyl-.beta.-D-thiogalactopyranoside (IPTG) or the like may be
added to the media when microorganisms transformed with the
expression vectors containing lac promoter are cultured;
indoleacrylic acid (IAA) or the like may be added when
microorganisms transformed with the expression vectors containing
trp promoter are cultured.
The media for culturing a transformant obtained by using an animal
cell as a host cell include a generally used RPMI1640 medium [The
Journal of the American Medical Association, 199, 519 (1967)],
Eagle's MEM medium [Science, 122, 501 (1952)], DMEM medium
[Virology, 8, 396 (1959)], 199 medium [Proceeding of the Society
for the Biological Medicine, 73, 1 (1950)] or those to which fetal
calf serum or the like is added.
Normally, the transformant is cultured in the presence of 5%
CO.sub.2 for 1 to 7 days at pH 6 to 8 and 30 to 40.degree. C.
If necessary, antibiotics e.g., kanamycin and penicillin may be
added to the medium while culturing.
Examples of media to culture a transformant obtained by using an
insect cell as a host cell include a generally used TNM-FH medium
(Pharmingen), Sf-900 II SFM medium (GIBCO BRL), ExCell400,
ExCell405 (both manufactured by JRH Biosciences), Grace's Insect
Medium (Grace, T.C.C., Nature, 195, 788 (1962)).
The transformant is generally cultured for 1 to 5 days at pH 6 to 7
and at 25.degree. C. to 30.degree. C.
If necessary, antibiotics e.g., gentamycin may be added to the
medium while culturing.
Proteins having activity to improve efficiency in the biosynthesis
of isoprenoid compounds of this invention can be isolated and
purified from the culture of the transformant of this invention by
standard isolation and purification techniques for a enzyme.
For example, when the protein of this invention is expressed in a
soluble form within the cell, after the culture is completed the
cells are recovered by centrifugation, suspended in aqueous buffer,
then disrupted using an ultrasonicator, french press, Manton Gaulin
homogenizer, Dyno-Mill, or the like, thereby obtaining cell-free
extracts. The cell-free extract is separated by centrifugation to
obtain the supernatant. The purified sample can be obtained from
the supernatant by one of or a combination of standard techniques
for isolating and purifying enzymes. Such techniques include a
solvent extracting technique, salting out technique using ammonium
sulfate, desalting technique, precipitation technique using organic
solvents, anion exchange chromatography using resins such as
diethylaminoethyl (DEAE)-Sepharose, and DIAION HPA-75 (Mitsubishi
Chemical Corp.), cation exchange chromatography using resins e.g.,
S-Sepharose FF (Pharmacia), hydrophobic chromatography using resins
e.g., butylsepharose, phenylsepharose, gel filtration using
molecular sieve, affinity chromatography, chromatofocusing, and
electrophoresis such as isoelectric focusing.
When the proteins that form inclusion bodies are expressed in the
cells, the cells are recovered, disrupted, and separated by
centrifugation, thereby obtaining precipitated fractions. From the
resulting precipitated fractions, the protein is recovered by
standard techniques, and then the insoluble protein is solubilized
using a protein denaturing agent. The solubilized solution is
diluted or dialyzed to an extent that the solution contains no
protein denaturing agent or that the concentration of protein
denaturing agent does not denature protein, thereby allowing the
protein to form a normal three-dimensional structure. Then the
purified sample can be obtained by the same techniques for
isolation and purification as described above.
When the protein of this invention or its derivative, such as a
sugar-modified protein, is secreted outside the cell, the protein
or its derivative, such as a sugar chain adduct, can be recovered
from the culture supernatant. That is, the culture is treated by
centrifugation and the like as described above so as to obtain
soluble fractions. From the soluble fractions, the purified sample
can be obtained using the techniques for isolation and purification
as described above.
The resulting protein as described above is, for example a protein
having an amino acid sequence selected from amino acid sequences of
SEQ ID NOS:1 to 5.
Moreover, the protein expressed by the method above can be
chemically synthesized by techniques including Fmoc method
(fluorenylmethyloxycarbonyl method), tBoc method
(t-butyloxycarbonyl method). Further, the protein can be
synthesized by using a peptide synthesizer of Souwa Boeki K.K.
(Advanced ChemTech, U.S.A), Perkin-Elmer Japan (Perkin-Elmer,
U.S.A), Pharmacia BioTech (Pharmacia BioTech, Sweden), ALOKA CO.,
LTD. (Protein Technology Instrument), KURABO INDUSTRIES LTD.
(Synthecell-Vega, U.S.A), PerSeptive Limited., Japan (PerSeptive,
U.S.A), or SHIMADZU CORP.
III. Production of Isoprenoid Compound
Isoprenoid compounds can be produced by culturing the transformants
obtained as described in II above according to the method of II
above, allowing the transformants to produce and accumulate
isoprenoid compounds in the culture, then recovering the isoprenoid
compounds from the culture.
The above culture can yield isoprenoid compounds, such as
ubiquinone, vitamin K.sub.2, and carotenoids. Specific examples of
isoprenoid compounds include ubiquinone-8 and menaquinone-8
produced using microorganisms belonging to the genus Escherichia as
a transformant, ubiquinone-10 produced using those belonging to the
genus Rhodobacter, vitamin K.sub.2 produced using those belonging
to the genus Arthrobacter as a transformant, astaxanthin produced
using those belonging to the genus Agrobacterium as a transformant,
and lycopene, .beta.-carotene, and zeaxanthin produced using those
belonging to the genus Erwinia as a transformant.
After the culture is completed, in order to isolate and purify
isoprenoid compounds, isoprenoid compounds are extracted by adding
an appropriate solvent to the culture, the precipitate is removed
by e.g., centrifugation, and then the product is subjected to
various chromatography.
IV. Screening a Substance Inhibiting Enzymatic Activity on
Non-Mevalonate Pathway
(1) Determination of Enzymatic Activity on Non-Mevalonate
Pathway
The enzymatic activity on non-mevalonate pathway can be determined
according to normal methods for determining enzymatic activity.
The pH of the buffer used as a reaction solution to determine
activity should be within a range that does not inhibit the
enzymatic activity of interest. A preferable pH range includes the
optimal pH.
For example, a buffer at pH 5 to 10, preferably 6 to 9 is used for
1-deoxy-D-xylulose 5-phosphate reductoisomerase.
Any buffer can be used herein so far as it does not inhibit the
enzymatic activity and can be adjusted to the pH above. Examples of
such a buffer include Tris-hydrochloric acid buffer phosphate
buffer, borate buffer, HEPES buffer, MOPS buffer, and bicarbonate
buffer. For example, Tris-hydrochloric acid buffer can preferably
be used for 1-deoxy-D-xylulose 5-phosphate reductoisomerase.
A buffer of any concentration may be employed so far as it does not
inhibit the enzymatic activity. The preferable concentration ranges
from 1 mol/l to 1 mol/l.
When the enzyme of interest requires a coenzyme, a coenzyme is
added to the reaction solution. For example, NADPH, NADH or other
electron donors can be used as a coenzyme for 1-deoxy-D-xylulose
5-phosphate reductoisomerase. A preferable coenzyme is NADPH.
Any concentration of the coenzyme to be added can be employed so
far as it does not inhibit reaction. Such a concentration
preferably ranges from 0.01 mol/l to 100 mol/l, more preferably,
0.1 mol/l to 10 mol/l.
Metal ions may be added to a reaction solution if necessary. Any
metal ion can be added so far as it does not inhibit reaction.
Preferable metal ions include Co.sup.2+, Mg.sup.2+, and
Mn.sup.2+.
Metal ions may be added as metallic salts. For example, a chloride,
a sulfate, a carbonate, and a phosphate can be added.
Any concentration of the metal ion to be added can be employed so
far as it does not inhibit reaction. A preferable concentration
ranges from 0 mol/l to 100 mol/l, more preferably, 0.1 mol/l to 10
mol/l.
The substrate of the enzyme of interest is added to the reaction
solution. For example, 1-deoxy-D-xylulose 5-phosphate is added for
1-deoxy-D-xylulose 5-phosphate reductoisomerase.
Any concentration of the substrate may be employed so far as it
does not inhibit reaction. The preferable concentration ranges from
0.01 mol/l to 0.2 mol/l in the reaction solution.
The enzyme concentration used in reaction is not specifically
limited. Normally, the concentration ranges from 0.01 mg/ml to 100
mg/ml.
An enzyme used herein is not necessarily purified into a single
substance. It may contain contaminative proteins. In the search as
described in (2) below, cellular extracts containing
1-deoxy-D-xylulose 5-phosphate reductoisomerase activity or cells
having the same activity can be used.
Any reaction temperature may be employed so far as it does not
inhibit enzymatic activity. A preferable temperature range includes
the optimal temperature. That is, the reaction temperature ranges
from 10.degree. C. to 60.degree. C., more preferably, 30.degree. C.
to 40.degree. C.
Activity can be detected by a method for measuring a decrease in
substrates accompanying the reaction or an increase in reaction
products as the reaction proceeds.
Such a method is a method wherein the substance of interest is
separated and quantitatively determined by e.g, high performance
liquid chromatography (HPLC) if necessary. When NADH or NADPH
increases or decreases as the reaction proceeds, activity can
directly be determined by measuring the absorbance at 340 nm of the
reaction solution. For example, the activity of 1-deoxy-D-xylulose
5-phosphate reductoisomerase can be detected by measuring a
decrease in the absorbance at 340 nm using a spectrophotometer to
determine NADPH quantity that decreases as the reaction
proceeds.
(2) Screening a Substance Inhibiting Enzymatic Activity on the
Non-Mevalonate Pathway
A substance inhibiting enzymatic activity on the non-mevalonate
pathway can be screened for by adding the substance to be screened
for to the enzymatic activity measurement system as described in
(1) above, allowing the mixture to react similarly, and then
screening a substance that suppresses the amount of the substrates
decreased in comparison to a case when no such substance is added;
or a substance that suppresses the yield of the reaction
product.
Screening methods include a method wherein the decrease in the
amount of substrates or the increase in the amount of reaction
products is traced with time; or a method where after the reaction
has proceeded for a certain period the decrease in the amount of
substrates or the increase in the amount of reaction products is
measured.
In the method wherein the decrease in the amount of substrates or
the increase in the amount of reaction products is traced with
time, the amount is measured preferably at 15 seconds to 20 minutes
intervals, mere preferably at 1 to 3 minutes intervals during
reaction.
To measure the decrease in the amount of substrates or the increase
in the amount of reaction products after reaction has proceeded for
a certain period, the reaction period is preferably 10 minutes to 1
day, more preferably, 30 minutes to 2 hours.
A substance inhibiting the enzymatic activity on the non-mevalonate
pathway inhibits the growth of microorganisms and plants that
possess the non-mevalonate pathway. The inventors have first found
the fact that this substance inhibits the growth of the
microorganisms and plants.
The non-mevalonate pathway is present in microorganisms and plants,
but absent in animals and humans. Therefore, the substance
inhibiting the enzymatic activity on the non-mevalonate pathway but
not affecting human and animals can be obtained by the above
described screening method.
This substance can be an effective antibiotic or herbicide.
This specification includes part or all of the contents as
disclosed in the specification and/or drawings of Japanese Patent
Application Nos. 10-103101, 10-221910 and 11-035739, which are
priority documents of the present application.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows the effect of reaction temperature on
1-deoxy-D-xylulose 5-phosphate reductoisomerase activity.
FIG. 2 shows the effect of the pH of the reaction solution on
1-deoxy-D-xylulose 5-phosphate reductoisomerase activity. Enzymatic
activity measured at various pH in 100 mol/l Tris-hydrochloric acid
buffer are shown. Activity is shown as a relative activity when
activity at pH 8.0 is considered as 100%.
FIG. 3 shows a method for disrupting yaeM gene on a chromosome
using homologous recombination.
FIG. 4 shows the effect of fosmidomycin on 1-deoxy-D-xylulose
5-phosphate reductoisomerase.
BEST MODE FOR CARRYING OUT THE INVENTION
The invention will now be described by way of examples, but shall
not be limited thereto. Unless otherwise specified, gene
recombination shown in the examples was carried out according to
techniques described in Molecular Cloning, Second Edition
(hereinafter referred to as the standard techniques).
EXAMPLE 1
Cloning of DNA Encoding Proteins Involved in the Biosynthesis of
Isoprenoid Compounds
(1) Cloning of DNA Encoding Proteins Involved in the Biosynthesis
of Isoprenoid Compounds Using the Nucleotide Sequence of E. coli
DXS Gene
One platinum loop of E. coli XL1-Blue (purchased from TOYOBO) was
inoculated into 10 ml of LB liquid medium, then cultured overnight
at 37.degree. C.
After culturing, cells were collected by centrifugation from the
resultant culture.
Chromosomal DNA was isolated and purified from the cells according
to the standard techniques.
Sense and antisense primers, each having BamHI and EcoR I
restriction enzyme sites at their 5'-ends and consisting of
nucleotide sequence pairs of SEQ ID NOS:12 and 13, 14 and 15, 12
and 16, 17 and 18, and 19 and 13; and sense and antisense primers,
each having BamHI restriction enzyme site at their 5'-ends and
consisting of a nucleotide sequence pair of SEQ ID NO:22 and 23;
were synthesized using a DNA synthesizer.
PCR was carried out with a DNA Thermal Cycler (Perkin Elmer
Instruments, Inc. Japan) using these primers, chromosomal DNA as a
template, and a TaKaRa La-PCR.TM. Kit Ver. 2 (TAKARA SHUZO CO.,
LTD.), Expand.TM. High-Fidelity PCR System (Boehringer Manheim
K.K.) or a Taq DNA polymerase (Boehringer).
PCR was carried out for 30 cycles. In the case of amplifying a DNA
fragment of 2 kb or less, one cycle consisting of reaction at
94.degree. C. for 30 seconds, 55.degree. C. for 30 seconds to 1
minute, and 72.degree. C. for 2 minutes; in the case of amplifying
a DNA fragment of more than 2 kb, one cycle consisting of reaction
at 98.degree. C. for 20 seconds, and 68.degree. C. for 3 minutes;
then followed by the reaction at 72.degree. C. for 7 minutes.
Among the DNA fragments amplified by PCR, DNA fragments amplified
using sense and antisense primers, each having BamH I and EcoR I
restriction enzyme sites at their 5'-ends, were digested with
restriction enzymes BamH I and EcoR I; DNA fragments amplified
using sense and antisense primers, each having BamH I restriction
enzyme site at their 5'-ends, were digested with restriction enzyme
BamH I.
After the digestion, these DNA fragments treated with the
restriction enzymes were subjected to agarose gel electrophoresis
and recovered BamH I and EcoR I-treated DNA fragments and BamH
I-treated DNA fragments.
A broad host range vector pEG 400 containing lac promoter [J. Bac.,
172, 2392 (1990)] was digested with restriction enzymes BamH I and
EcoR I, subjected to agarose gel electrophoresis and recovered BamH
I and EcoR I-treated pEG 400 fragments.
pUC118 (TAKARA SHUZO CO., LTD.) was digested with a restriction
enzyme BamH I, then subjected to agarose gel electrophoresis and
recovered BamH I-treated pUC 118 fragments.
Each of the resultant BamH I and EcoR I-treated DNA fragments was
mixed with BamH I and EcoR I-treated pEG 400 fragments, then the
mixture was allowed to precipitate with ethanol. The obtained DNA
precipitate was dissolved in 5 .mu.l of distilled water for
ligation reaction to occur, thereby obtaining each recombinant
DNA.
Using the resultant recombinant DNA, E. coli (purchased from
TOYOBO) DH5 .alpha. was transformed according to the standard
techniques. Then the transformant was spread on LB agar medium
containing 100 .mu.g/ml of spectinomycin, then cultured overnight
at 37.degree. C.
Some colonies of the transformant resistant to spectinomycin were
cultured in 10 ml of LB liquid medium containing 100 .mu.g/ml of
spectinomycin with shaking for 16 hours at 37.degree. C.
The resulting culture was centrifuged, so that cells were
collected.
Plasmids were isolated from the cells according to the standard
techniques.
To confirm that the isolated plasmids contained the DNA fragment of
interest, the plasmids were cleaved with various restriction
enzymes to examine their structures and their nucleotide sequences
were sequenced.
A plasmid containing a DNA with a nucleotide sequence of SEQ ID
NO:6, DNA with a nucleotide sequence of SEQ ID NO:7, DNA with a
nucleotide sequence of SEQ ID NO:8, and DNA with a nucleotide
sequence of SEQ ID NO:9 was named pADO-1. A plasmid containing a
DNA with a nucleotide sequence of SEQ ID NO:6 was named pDXS-1. A
plasmid containing a DNA with a nucleotide sequence of SEQ ID NO:7
was named pISP-1. A plasmid containing a DNA with a nucleotide
sequence of SEQ ID NO:9 was named pTFE-1.
The above BamH I-treated DNA fragments and BamH I-treated pUC118
fragments were mixed, then the mixture was allowed to precipitate
with ethanol. The resulting DNA precipitate was dissolved in 5
.mu.l of distilled water for ligation reaction to occur to obtain
recombinant DNA. Escherichia coli was transformed using the
recombinant DNA in the same manner as described above, then
plasmids were isolated from the transformants.
To confirm the isolated plasmids contain the DNA fragments of
interest, the plasmids were cleaved with various restriction
enzymes to examine their structures and their nucleotide sequences
were sequenced in the same manner as described above.
These plasmids were digested with BamH I. The DNA fragments of
interest were recovered in the same manner as described above, then
sub-cloned into an expression vector pQE30 (Qiagen, Inc).
The plasmid obtained by the sub-cloning above and having a
nucleotide sequence of SEQ ID NO:6 was named pQEDXS-1.
(2) Cloning of the Gene Complementing Methylerythritol-requiring
Nature
Selection of Methylerythritol-requiring Mutant of Escherichia
coli
E. coli W3110 (ATCC 14948) was inoculated into LB liquid medium and
cultured to its logarithmic growth phase.
After culturing, cells were recovered from the resulting culture by
centrifugation.
The cells were washed with 0.05 mol/l Tris-maleate buffer (pH 6.0),
then suspended in the same buffer to the cell density of 10.sup.9
cells/ml.
Mutation was induced by adding NTG to the suspension to a final
concentration of 600 mg/l, and then the mixture was maintained for
20 minutes at room temperature.
These NTG treated cells were spread on M9 minimal agar medium
containing 0.1% methylerythritol (Molecular Cloning, Second
Edition) plate and cultured.
Methylerythritol was chemically synthesized according to the method
described in Tetrahedron Letters, 38, 35, 6184 (1997).
Colonies grown on M9 minimal agar medium containing 0.1%
methylerythritol were replicated on M9 minimal agar medium and on
M9 minimal agar medium containing 0.1% methylerythritol. The mutant
of interest, a strain requiring methylerythritol to grow, was
selected. That is, a strain capable of growing on a minimal agar
medium containing 0.1% methylerythritol but not on the same lacking
methylerythritol was selected.
The thus obtained methylerythritol-requiring mutant ME7 was used in
the following experiments.
Cloning of the Gene Complementing Methylerythritol-requiring
Nature
Escherichia coli W3110 (ATCC14948) was inoculated into LB liquid
medium, then cultured to its logarithmic growth phase. Then cells
were collected from the resultant culture by centrifugation.
Chromosomal DNA was isolated and purified from the obtained cells
according to the standard techniques.
200 .mu.g of the chromosomal DNA was partially digested with a
restriction enzyme, Sau 3AI. The resulting DNA fragments were
fractionated by sucrose density-gradient centrifugation (26,000
rpm, 20.degree. C., 20 hr).
The DNA fragments obtained by the above fractionation, 4 to 6 kb
each, were ligated to pMW118 vector (Nippon Gene), which had been
digested with a restriction enzyme BamH I, constructing a genomic
DNA library.
Using this genomic DNA library, the strain ME7 isolated in above
was transformed according to the standard techniques.
The resulting transformants were spread on LB agar medium
supplemented with 100 .mu.g/l of ampicillin, then cultured
overnight at 37.degree. C.
Plasmids were extracted from each colony that grew on the agar
medium and then the nucleotide sequences were determined.
The plasmids determined its nucleotides sequence had contained the
nucleotide sequence of SEQ ID NO:10. These plasmids were named
pMEW41 and pMEW73.
A plasmid extracted from one strain of the clones having the
sequence was named pMEW73.
The pMEW73 was double-digested with Hind III and Sac I. The
resultant Hind III and Sac I-treated DNA fragment having a
nucleotide sequence of SEQ ID NO:10 was ligated to multi-cloning
sites of broad host range vector pEG400 [J. Bac., 172, 2392
(1990)], constructing pEGYM1.
The Hind III-Sac I-treated DNA fragment was ligated to the Hind
III-Sac I site of vector pUC19 (Gene, 33, 103 (1985)), constructing
pUCYM-1.
According to the information on the nucleotide sequence of
chromosomal DNA of Escherichia coli based on Genbank data base, the
DNA fragment that had been inserted into the vector was confirmed
to contain yaeM gene.
A recombinant vector, which can express yaeM gene sufficiently, was
constructed by following method with PCR [Science, 230, 1350
(1985)].
A sense primer having a sequence of SEQ ID NO:20 and an antisense
primer having a sequence of SEQ ID NO:21 were synthesized using a
DNA synthesizer.
A Bam H I restriction enzyme recognition site was added to each
5'-end of the sense and antisense primers.
yaeM gene was amplified by PCR with DNA Thermal Cycler (Perkin
Elmer Instruments, Inc. Japan) using chromosomal DNA of E. coli as
a template, these primers and Taq DNA polymerase (Boelinnger).
PCR was carried out by 30 cycles, one cycle consisting of reaction
at 94.degree. C. for 30 seconds, reaction at 55.degree. C. for 30
seconds, and reaction at 72.degree. C. for 2 minutes followed by
reaction at 72.degree. minutes.
After the amplified DNA fragments and pUC118 (TAKARA SHUZO CO.,
LTD.) were digested with a restriction enzyme BamH I, each of the
DNA fragments were purified by agarose gel electrophoresis.
Both of these fragments were mixed, then the mixture was allowed to
precipitate with ethanol. The resultant DNA precipitate was
dissolved in 5 .mu.l of distilled water for ligation reaction to
occur, thereby obtaining recombinant DNA.
The recombinant DNA was confirmed to be yaeM gene by determining
the nucleotide sequences, then sub-cloned to expression vector
pQE30 (Qiagen, Inc).
The resulting recombinant DNA was named pQEYM1.
The strain ME7 was transformed using pQEYM1 by standard techniques.
The transformant was spread on LB agar medium containing 100
.mu.g/ml of ampicillin, then cultured overnight at 37.degree. C.
The transformants were confirmed to form colonies at the same
growth rate as wild-type strain, suggesting that yaeM gene
complemented mutation in the strain ME7.
EXAMPLE 2
Production of Ubiquinone-8 (CoQ8) Using Recombinant Escherichia
coli
(1) E. coli DH5.alpha. were transformed using the plasmids pADO-1,
pDXS-1, and pXSE-1, those obtained in Example 1 above, and pEG400
as a control, respectively, then E. coli DH5 .alpha./pAD0-1, E.
coli DH5 .alpha./pDXS-1, E. coli DH5 .alpha./pXSE-1 and E. coli DH5
.alpha./pEG400 that showed resistance to spectinomycin at a
concentration of 100 .mu.g/ml were obtained.
These transformants were inoculated into a test tube containing 10
ml of LB medium supplemented with thiamine and vitamin B.sub.6, 100
mg/l each, 50 mg/l of p-hydroxybenzoic acid, and 100 .mu.g/ml of
spectinomycin. Then the transformants were cultured with shaking
for 72 hours at 30.degree. C.
After the culture was completed, each culture was concentrated
10-fold.
To each 300 .mu.l of concentrated culture, 300 .mu.l 2-butanol and
300 .mu.L glass beads were added. Isoprenoid compounds were
extracted with the solvent while disrupting the cells by Multi
Beads Shocker MB-200 (YASUI KIKAI) for 5 minutes. Then the
2-butanol layer was collected by centrifugation.
The amount of CoQ8 produced by the transformants was calculated by
Quantitative analysis of the CoQ8 in the butanol layer using high
performance liquid chromatography (LC-10A, SHIMADZU CORP.).
HPLC was carried out using Develosil ODS-HG-5 (NOMURA CHEMICAL
K.K.) as a column, and methanol:n-hexane=8:2 solution as a mobile
phase at 1 ml/min of the flow rate and 275 nm of the measuring
wavelength.
Table 1 shows the results.
TABLE-US-00001 TABLE 1 CoQ8 Production by transformant of
Escherichia coli Cell Amount Amount of CoQ8 Intracellular
Transformant (OD660) Produced (mg/L) Content.sup.1 E. coli 5.8 0.63
1.1 DH5 .alpha./pEG400 E. coli 5.5 0.98 1.8 DH5 .alpha./pADO-1 E.
coli 5.2 0.85 1.6 DH5 .alpha./pDXS-1 E. coli 5.6 0.67 1.2 DHS
.alpha./pXSE-1 *.sup.1Intracellular content is shown with a value
obtained by dividing a 10-fold CoQ8 production (mg/L) by a cell
amount (OD660). The amount of CoQ8 produced was significantly
higher in DH5 .alpha./pADO-1, DH5 .alpha./pDXS-1 and DH5
.alpha./pXSE-1 than in the control strain DH5 .alpha./pEG400. In
particular, the highest productivity was shown by DH5
.alpha./pADO-1 to which all DNA obtained in Example 1 were
introduced. (2) E. coli DH5 .alpha./pDXS-1 or E. coli DHS
.alpha./pEG400, as obtained in (1) above, was inoculated into a
test tube containing 10 ml of a M9 medium, and then cultured with
shaking for 72 hours at 30.degree. C.
After the culture was completed, the amount of CoQ8 produced by the
transformants was calculated in the same manner as in (1)
above.
Table 2 shows the results.
TABLE-US-00002 TABLE 2 CoQ8 Production by transformant of
Escherichia coli Cell Amount Amount of CoQ8 Intracellular
Transformant (OD660) Produced (mg/L) Content*.sup.1 E. coli 3.1
0.49 1.6 DH5 .alpha./EG400 E. coli 2.5 1.02 4.1 DH5 .alpha./pDXS-1
*.sup.1Intracellular content is shown with a value obtained by
dividing a 10-fold CoQ8 production (mg/L) by a cell amount (OD660).
The amount of CoQ8 produced was significantly higher in DH5
.alpha./pDXS-1 than in the control strain DH5 .alpha./pEG400. (3)
Production of CoQ8 using Recombinant Escherichia coli
The plasmid pEGYM1 obtained in Example 1 or pEG400 as a control was
introduced into E. coli DH5 .alpha. and E. coli DH5 .alpha./pEGYM1
and E. coli DH5 .alpha./pEG400 that show resistance to
spectinomycin at a concentration of 100 .mu.g/ml were obtained.
These transformants were inoculated into a test tube containing 10
ml of LB medium supplemented with 1% glucose, 100 mg/l of vitamin
B.sub.1, 100 mg/l of vitamin B.sub.6, 50 mg/l of p-hydroxybenzoic
acid. Then the transformants were cultured with shaking for 72
hours at 30.degree. C.
After the culture was completed, the amount of CoQ8 produced by the
transformants was calculated in the same manner as in (1)
above.
Table 3 shows the results.
TABLE-US-00003 TABLE 3 CoQ8 Production by transformants of
Escherichia coli Cell Amount Amount of CoQ8 Intracellular
Transformant (OD660) Produced (mg/L) Content*.sup.1 E. coli 14.44
0.83 0.57 DH5 .alpha./pEG400 E. coli 13.12 0.94 0.71 DH5
.alpha./pEGYM1 *.sup.1Intracellular content is shown with a value
obtained by dividing a 10-fold CoQ8 production (mg/L) by a cell
amount (OD660). The amount of CoQ8 produced was significantly
higher in DH5 .alpha./pEGYM1 than in the control strain DH5
.alpha./pEG400.
EXAMPLE 3
Production of Menaquinone-8 (MK-8) by Recombinant Escherichia
coli
(1) The E. coli DH5 .alpha./pADO-1 or E. coli DH5 .alpha./pEG400,
obtained in Example 2 (1), inoculated into a test tube containing
10 ml of TB medium supplemented with 100 .mu.g/ml of spectinomycin,
and then cultured with shaking for 72 hours at 30.degree. C. The TB
medium had been prepared by dissolving 12 g of bactotrypton
(Difco), 24 g of yeast extract (Difco), and 5 g of glycerol into
900 ml of water followed by the addition of 100 ml of aqueous
solution containing 0.17 mol/l KH.sub.2PO.sub.4 and 0.72 mol/l
K.sub.2HPO.sub.4.
After the culture was completed, MK-8 was quantified in the same
quantifying method for CoQ8 as in Example 2 (1), then the amount of
MK-8 produced by the transformants was calculated.
Table 4 shows the results.
TABLE-US-00004 TABLE 4 MK-8 Production by transformants of
Escherichia coli Cell Amount Amount of MK-8 Intracellular
Transformant (OD660) Produced (mg/L) Content*.sup.1 E. coli 23.2
1.1 0.46 DH5 .alpha./pEG400 E. coli 23.5 1.8 0.75 DH5 .alpha./ADO-1
*.sup.1Intracellular content is shown with a value obtained by
dividing a 10-fold CoQ8 production amount (mg/L) by a cell amount
(OD660). The amount of MK-8 produced was significantly higher in
DH5 .alpha./pADO-1 than in the control DH5 .alpha./pEG400. (2) E.
coli DH5 .alpha./pDXS-1 or E. coli DH5 .alpha./pEG400, obtained in
Example 2 (1), was cultured in the same manner in (1) above, then
the amount of MK-8 produced by the transformants was
calculated.
Table 5 shows the results.
TABLE-US-00005 TABLE 5 Production of MK-8 by transformants of
Escherichia coli Cell Amount Amount of MK-8 Intracellular
Transformant (OD660) Produced (mg/L) Control*.sup.1 E. coli 42.8
2.41 0.56 DHS .alpha./pEG400 E. coli 44.0 2.96 0.67 DH5
.alpha./pDXS-1 *.sup.1Intracellular content is shown with a value
obtained by dividing a 10-fold CoQ8 production (mg/L) by a cell
amount (OD660). The amount of MK-8 produced was significantly
higher in DH5 .alpha./pDXS-1 than in the control strain DH5
.alpha./pEG400.
EXAMPLE 4
Production of CoQ8 by Recombinant Erwinia carotovora
A plasmid pDXS-1 obtained in Example 1 or pEG400 as a control, was
introduced into Erwinia carotovora IFO-3380, thereby obtaining
transformants IFO-3380/pDXS-1 and IFO-3380/pEG400, both of which
were resistant to spectinomycin at a concentration of 100
.mu.g/ml.
These transformants were inoculated into a test tube containing 10
ml of LB medium supplemented with 100 .mu.g/ml of spectinomycin,
and then cultured with shaking for 72 hours at 30.degree. C.
After the culture was completed, the amount of CoQ8 produced by the
transformants was calculated in the same manner as in Example 2
(1).
Table 6 shows the results.
TABLE-US-00006 TABLE 6 CoQ8 Production by transformants of Erwinia
carotovora Cell Amount Amount of CoQ8 Intracellular Transformant
(OD660) Produced (mg/L) Content*.sup.1 IFO-3380/Peg400 1.68 0.26
1.5 Ifo-3380/Pdxs-1 2.48 0.45 1.8 *.sup.1Intracellular content is
shown with a value obtained by dividing a 10-fold CoQ8 production
(mg/L) by a cell amount (OD660). The amount of CoQ8 produced was
significantly higher in IFO-3380/pDXS-1 than in the control strain
IFO-3380/pEG400.
EXAMPLE 5
Production of Ubiquinone and Carotenoids by Recombinant Erwinia
uredovora
The plasmids pUCYM-1, pQEDXS-1, pQEYM-1, obtained in Example 1, or
pUC19 and pQE30 as controls were introduced into Erwinia uredovora
DSM-30080 by electroporation, and then the transformants, E.
uredovora DSM-30080/pUCYM-1, E. uredovora DSM-30080/pQEDXS-1, E.
uredovora DSM-30080/pQEYM-1, E. uredovora DSM-30080/pUC19 and E.
uredovora DSM-30080/pQE30, which showed resistant to ampicillin at
a concentration of 100 .mu.g/ml were obtained.
These transformants were inoculated into a test tube containing 10
ml of LB medium supplemented with 100 .mu.g/ml of ampicillin, 1%
glucose, vitamin B.sub.1 and vitamin B.sub.6, 100 mg/l each, and 50
mg/l of p-hydroxybenzoic acid. Then the transformants were cultured
by shaking for 72 hours at 30.degree. C.
After the culture was completed, the amount of CoQ8 produced by the
transformants was calculated in the same manner as in Example 2
(1).
The produced amount of carotenoid pigments was calculated by
detecting the absorbance at 450 nm for the 2-butanol layer using a
spectrophotometer in the same manner as in Example 2 (1).
Table 7 shows the results.
TABLE-US-00007 TABLE 7 Production of CoQ8 and Carotenoids by
transformants of E. uredovora CoQ8 Carotenoids Intracellular
Intracellular Cell content ratio Production content ratio amount
Production Relative Relative Relative Transformants OD660 mg/L
value value value DSM-30080/pUC19 2.00 1.15 1.0 1.0 1.0
DSM-30080/pUCYM-1 1.88 1.39 1.3 1.5 1.6 DSM-30080/pQE30 2.52 1.29
1.0 1.0 1.0 DSM-30080/pQEYM-1 1.92 1.36 1.4 1.7 2.2
DSM-30080/pQEDXS-1 2.12 3.21 3.0 5.6 6.7
Both CoQ8 production and carotenoid pigment production were
significantly higher in DSM-30080/pUCYM-1 than in the control
strain DSM-30080/pUC19.
Similarly, both CoQ8 production and carotenoid pigment production
were significantly higher in DSM-30080/pQEYM-1 and
DSM-30080/pQEDXS-1 than in the control strain DSM-30080/pQE30.
EXAMPLE 6
Cloning of the DNA Encoding Proteins Involved in the Biosynthesis
of Isoprenoid Compounds From a Photosynthetic Bacterium Rhodobacter
sphaeroides
(1) Cloning of DXS Gene From R. sphaeroides
The Genbank database was searched for DXS homologue conserved in
other species using the DXS nucleotide sequence found in E. coli.
As a result, DXS homologues were found in Haemophilus influenzae
(P45205), Rhodobacter capsulatus (P26242), Bacillus subtilis
(P54523), Synechocystis sp. PCC6803 (P73067) and Mycobacterium
tuberculosis (007184) and the like. Highly conserved amino acid
sequences were selected by comparison of these sequences. A
nucleotide sequence corresponding to such a conserved amino acid
sequence was designed in consideration of the codon usuage in R.
sphaeroides. A DNA fragment having a nucleotide sequence of SEQ ID
NO:32 and of SEQ ID NO:33, and a DNA fragment having a nucleotide
sequence of SEQ ID NO:34 were synthesized by DNA synthesizer.
PCR was carried out with DNA Thermal Cycler (Perkin Elmer
Instruments, Inc. Japan) using chromosomal DNA of R. sphaeroides
KY4113 (FERM-P4675) as a template, the primers above, and an
Expand.TM. High-Fidelity PCR System (Boehringer Manheim K.K.).
PCR was carried out by 30 cycles, one cycle consisting of reaction
at 94.degree. C. for 40 seconds, reaction at 60.degree. C. for 40
seconds, reaction at 72.degree. C. for 1 minute, followed by
reaction at 72.degree. C. minute, thereby obtaining the DNA
fragment of interest. The DNA fragments were DIG-labeled using DIG
DNA Labeling Kit (Boehringer Manheim K.K.).
To obtain the full length DXS gene of R.sphaeroides, a genomic DNA
library of a strain KY4113 was constructed. The strain KY4113 was
cultured overnight in LB medium, extracting the chromosomal DNA.
The chromosomal DNA was partially digested with a restriction
enzyme Sau3AI, and then 4 to 6 kb DNA fragments were purified by
sucrose density-gradient centrifugation. The DNA fragments were
ligated with BamtH I-digested vector pUC19 using a Ligation Pack
(Nippon Gene), and E. coli DH5.alpha. was transformed using the
ligated DNA. The transformants were spread on LB agar medium
containing 100 .mu.g/ml of ampicillin, thus obtaining about 10,000
colonies. As a result of screening by colony hybridization using
the DIG-labeled DNA fragment as a probe, which had been obtained by
the above method, two types of DNA fragments were detected. As a
result of sequencing, ORF sharing high degrees of sequence homology
with known DXS gene of other species was found from each DNA
fragment. An amino acid sequence of SEQ D NO:26 was named DXS1 and
that of SEQ ID NO:27 was named DXS2.
As a result of sequencing, ORF sharing high degrees of sequence
homology with known DXS gene of other species was found from each
DNA fragment. An amino acid sequence of SEQ ID NO:26 was named DXS1
and that of SEQ ID NO:27 was named DXS2.
(2) Confirmation of Complementarity Using E. coli DXS Gene-Deleted
Mutant
Selection of E. coli DXS Gene-Deleted Strain
E. coli W3110 (ATCC14948) was inoculated into LB liquid medium, and
then cultured to its logarithmic growth phase. After culturing,
cells were collected from the culture by centrifugation.
The cells were washed with 0.05 mol/l Tris-maleate buffer (pH 6.0)
and suspended in the same buffer to a cell density of 10.sup.9
cells/ml.
NTG was added to the suspension to a final concentration of 600
mg/l, then the mixture was maintained for 20 minutes at room
temperature to induce mutation.
The resultant NTG-treated cells were spread on a M9 minimum agar
medium (Molecular Cloning, Second Edition) plate containing 0.1%
1-deoxyxylulose, then cultured. 1-Deoxyxylulose had been chemically
synthesized according to the method described in J. C. S. Perkin
Trans I, 2131-2137 (1982).
Colonies grew on M9 minimum agar medium containing 0.1%
1-deoxyxylulose were replicated on M9 minimal agar medium and on M9
minimal agar medium containing 0.1% 1-deoxyxylulose. The mutant of
interest, a strain requiring 1-deoxyxylulose to grow, was selected.
That is, a strain capable of growing on minimal agar medium
containing 1-deoxyxylulose but not on the same medium lacking
1-deoxyxylulose was selected.
The thus selected and obtained mutant was named ME1.
When pDXS-1 was introduced into the strain ME1, deficiency in
1-deoxyxylulose of the strain ME1 was complemented. Therefore the
strain ME1 was confirmed to be a strain from which DXS gene was
deleted.
(3) Complementation Studies on DXS 1 and DXS2
DNA fragment encoding DXS1 of SEQ ID NO:27 or a DNA fragment
encoding DXS2 of SEQ ID NO:29, respectively, both derived from the
strain KY4113, was ligated to downstream of the lac promoter of a
vector pUC19 respectively to construct recombinant plasmids.
When the constructed plasmids were introduced into the strain ME1,
both DXS1 and DXS2 each complemented the 1-deoxyxylulose-deficiency
in the strain ME 1.
Therefore, R. sphaeroides was shown to have two genes, DXS1 and
DXS2, having activity to catalyze the reaction to produce
1-deoxy-D-xylulose 5-phosphate from pyruvic acid and glyceraldehyde
3-phosphate.
(4) Cloning of Gene Complementing Methylerythritol-requiring Nature
Derived From R. sphaeroides
The E. coli Methylerythritol-requiring mutant ME7 obtained in
Example 1(2) was inoculated into LB liquid medium containing 0.1%
methylerythritol, cultured to its logarithmic growth phase, then
centrifuged to collect cells.
The cells were washed twice with 1 mol/HEPES aqueous solution
containing 10% glycerol so as to remove the medium components as
far as possible.
Plasmids were extracted from the genomic library of R. sphaeroides
KY4113 constructed in Example 6 (1). Then the plasmids were
introduced into the washed cells by electroporation according to
standard techniques.
Next, the cells were spread on LB agar medium containing 100
.mu.g/l of ampicillin, then cultured overnight at 37.degree. C.
After picking up the colonies grown on the medium, the colonies
were inoculated into LB liquid medium to culture, then plasmids
were extracted from the cells cultured.
When the plasmids extracted were introduced again into the strain
ME 7, the transformants could grow in a medium lacking
methylerythritol. Therefore it was confirmed that the plasmid
contained a DNA fragment complementing methylerythritol-requiring
nature derived from R. sphaeroides.
As a result of sequencing of the nucleotide sequence of the DNA
fragment, the DNA sequence of SEQ ID NO:31 encoding an amino acid
sequence that shares high homology with E. coli yaeM was found.
EXAMPLE 7
Production of Ubiquinone-10 (CoQ10) By Recombinant Photosynthetic
Bacteria
A glnB promoter derived from the strain KY4113 was ligated upstream
of the DNA fragment DXS1 of SEQ ID NO:27 and DXS2 of SEQ ID NO:29,
both obtained in Example 6. Then the product was inserted into a
broad host range vector pEG400, thus constructing plasmids. These
plasmids were named pRSDX-1 and pRSDX-2, respectively. In addition,
yaeM and DXS1 were joined in tandem, then the product was ligated
downstream of ginB promoter, thereby constructing a plasmid. The
plasmid was named pRSYMDX1. These plasmids were introduced into R.
sphaeroides KY4113, respectively, by electroporation (Bio-Rad
Laboratories).
Then the cells were spread on LB agar medium containing
spectinomycin at a concentration of 100 .mu.g/ml, then cultured for
3 days at 30.degree. C.
Next, colonies that grew on the medium were inoculated into LB
medium containing spectinomycin at a concentration of 100 .mu.g/ml,
cultured ovenight. Then, the cultured cells were collected by
centrifugation.
It was confirmed that the cells of each strain contained the
introduced plasmid by extracting the plasmids from the cells
(Qiagen, Inc). Thus obtained transformants were named
KY4113/pRSDX-1, KY4113/pRSDX-2, KY4113/pRSYMDX1 and
KY4113/pEG400.
A platinum loop of each transformant was inoculated into a test
tube containing 5 ml of seed medium (2% glucose, 1% peptone, 1%
yeast extract, 0.5% NaCl, pH 7.2 adjusted with NaOH) and then
cultured for 24 hours at 30.degree. C.
0.5 ml of the resultant culture was inoculated into a test tube
containing 5 ml of ubiquinone-10 production medium, then cultured
by shaking for 5 days at 30.degree. C. The ubiquinone-10 production
medium consisted of 4% blackstrap molasses, 2.7% glucose, 4% corn
steep liquor, 0.8% ammonium sulfate, 0.05% potassium primary
phosphate, 0.05% potassium secondary phosphate, 0.025% magnesium
sulfate heptahydrate, 3 mg/l of ferrous sulfate heptahydrate, 8
mg/l of thiamine, 8 mg/l of nicotinic acid, and 1 ml/l of trace
element, had previously been adjusted to pH 9, supplemented with 1%
calcium carbonate, then autoclaved.
Then the amount of CoQ10 produced by the transformants was
calculated in the same manner as in quantification of CoQ8 in
Example 2 (1). Table 8 shows the results.
TABLE-US-00008 TABLE 8 Amount of CoQ10 Cell amount [OD660]
Accumulated [mg/l] KY4113/pEG400 23.7 65.2 KY4113/pRSDX-1 23 81
KY4113/pRSDX-2 24.4 81.9 KY4113/pRSYMDX1 25.8 117.9 The amount of
CoQ10 produced was significantly higher in KY4113/pRSDX-1,
KY4113/pRSDX-2 and KY4113/pRSYMDX1 than in the control strain
KY4113/pEG400.
EXAMPLE 8
Determination of the Activity of the Enzyme Encoded By yaeM
Gene
(1) Overexpression of yaeM Gene
A recombinant plasmid that can express yaeM gene sufficiently was
constructed using PCR [Science, 230, 1350 (1985)], as follows.
A sense primer having a nucleotide sequence of SEQ ID NO:24 and an
antisense primer having a nucleotide sequence of SEQ ID NO:25 were
synthesized using a DNA synthesizer.
A restriction enzyme BamH I site was added to each of 5'-ends of
the sense and antisense primers.
yaeM gene was amplified by PCR using chromosomal DNA of E. coli as
a template, these primers, Taq DNA polymerase (Boehringer), and DNA
Thermal cycler (Perkin Elmer Japan).
PCR was carried out by 30 cycles, one cycle consisting of reaction
at 94.degree. C. for 30 seconds, reaction at 55.degree. C. for 30
seconds, and reaction at 72.degree. C. for 2 minutes followed by
reaction at 72.degree. C. for 7 minutes.
The amplified DNA fragments and pUC118 (TAKARA SHUZO Co., Ltd.)
were digested with a restriction enzyme BamH I, then each DNA
fragment was purified by agarose gel electrophoresis.
Both purified fragments were mixed together, then treated with
ethanol, allowing DNA to precipitate. The resultant DNA precipitate
was dissolved in 5 .mu.l of distilled water for ligation reaction
to occur, thereby obtaining recombinant DNA.
The recombinant DNA was confirmed to be yaeM gene by determining
its DNA sequence.
Plasmids were extracted from the microorganism having the
recombinant DNA, digested with a restriction enzyme BamH I, and
subjected to agarose gel electrophoresis, thereby obtaining DNA
fragments containing BamH I-treated yaeM gene.
pQE30 (Qiagen, Inc) was digested with a restriction enzyme BamH I,
then subjected to agarose gel electrophoresis, thereby obtaining
BamH I-treated pQE30 fragments.
The resultant DNA fragments containing BamH I-treated yaeM gene
were mixed with BamH I-digested pQE30 fragments, and treated with
ethanol for DNA to precipitate. The DNA precipitate was dissolved
in 5 .mu.l of distilled water for ligation reaction to occur,
thereby obtaining recombinant DNA.
E. coli JM109 was transformed using the recombinant DNA by standard
techniques. Then the transformants were spread on LB agar medium
containing 100 .mu.g/ml of ampicillin, then cultured overnight at
37.degree. C.
Plasmids were isolated from the E. coli in the same manner as
described above.
Similarly, the isolated plasmid was cleaved with various
restriction enzymes to examine the structure, then the nucleotide
sequence was determined, thereby confirming the plasmids contained
the DNA fragments of interest. The plasmid was named pQEDXR.
(2) Determination of Activity of yaeM Gene Product
Purification of yaeM Gene Product
The pQEDXR constructed in (1) was introduced into E. coli M15
(Qiagen, Inc) having pREP4 by standard techniques, and a strain
M15/pREP4+pQEDXR resistant to 200 .mu.g/ml of ampicillin and 25
.mu.g/ml of kanamycin was obtained.
The strain M15/pREP4+pQEDXR was cultured at 37.degree. C. in 100 ml
of LB liquid medium containing 200 .mu.g/ml of ampicillin and 25
.mu.g/ml of kanamycin. When the turbidity at 660 nm reached 0.8,
isopropyl thiogalactoside was added to a final concentration of 0.2
mol/l. Subsequently, the strain was cultured for 5 hours at
37.degree. C., then the supernatant of the culture was removed by
centrifugation (3000 rpm, 10 minutes). The cells were suspended in
6 ml of 100 mol/l Tris-hydrochloric acid buffer (pH 8.0), then
disrupted using a ultrasonicator (SONIFIER, BRANSON) while cooling
with ice. The obtained cell-disrupted solution was centrifuged at
10,000 rpm for 20 minutes at 4.degree. C., thereby collecting the
supernatant. The supernatant centrifuged from the cellular extract
was introduced into a Ni-NTA resin column (Qiagen, Inc), then
washed with 20 ml of a washing buffer (100 mol/l Tris-hydrochloric
acid (pH 8.0), 50 mol/l imidazole, 0.5% Tween 20). Then 10 ml of an
elution buffer (100 mol/l Tris-hydrochloric acid (pH 8.0), 200
mol/l imidazole) was introduced into the column, thus fractionating
the eluate into 1 ml each.
Protein amounts for each fraction were measured using a kit for
quantifying protein amount (Bio-Rad Laboratories), thus obtaining a
fraction containing proteins as a purified protein fraction.
Preparation of a Substrate, 1-Deoxy-D-xylulose 5-Phosphate
A reaction substrate, 1-deoxy-D-xylulose 5-phosphate was prepared
as described below. 1-Deoxy-D-xylulose 5-phosphate was detected by
measuring the absorbance at 195 nm using HPLC [Column: Senshu pak
NH2-1251-N (4.6.times.250 mm, Senshu), mobile phase:100 mol/l
KH.sub.2PO.sub.4 (pH 3.5)].
The plasmid pQDXS-1 that allows overexpression of E. coli dxs gene
was introduced into E. coli M15/pREP4 in the same manner as
described above, obtaining a strain M15/pREP4+pQDXS-1.
This strain was cultured in the same way as in Example 8 (2) , then
dxs protein was purified using Ni-NTA resin column.
The purified dxs protein was added to a 20 ml of reaction solution
[100 mol/l Tris-hydrochloric acid (pH 7.5), 10 mol/l sodium
pyruvate, 30 moli DL-glyceraldehyde-3-phosphate, 1.5 mol/l thiamine
pyruvate, 10 mol/l MgCl.sub.2, 1 mol/l DL-dithiothreitol] then
maintained at 37.degree. C.
After reacting for 12 hours, the reaction solution was diluted with
water to 300 ml, introduced into an activated carbon column
(2.2.times.8 cm) followed by a Dowex 1-X8 (C1-type, 3.5.times.25
cm), then eluted with 1% saline solution. After the eluted fraction
was concentrated, the fraction was introduced into Sephadex G-10
(1.8.times.100 cm), then eluted with water. Finally fractions
containing 1-deoxy-D-xylulose 5-phosphate were freeze-dried,
thereby obtaining about 50 mg of white powder.
This powder was confirmed to be 1-deoxy-D-xylulose 5-phosphate by
NMR analysis (A-500, JEOL Ltd.).
Determination of Enzymatic Activity of yaeM Gene Product
0.3 mol/l of 1-deoxy-D-xylulose 5-phosphate (final concentration)
synthesized as described above was added to 1 ml of a reaction
solution containing 100 mol/l Tris-hydrochloric acid (pH 7.5), 1
mol/l MmCl.sub.2, 0.3 mol/l NADPH and yaeM gene product obtained in
Example 8 (2) , and then incubated at 37.degree. C. The increase
and decrease in NADPH during incubation was traced by reading the
absorbance at 340 nm using a spectrophotometer (UV-160, SHIMADZU
CORP.), suggesting that NADPH decreased with time.
To confirm the structure of the reaction product, the reaction was
carried out similarly, but on a larger scale, thus isolating the
product. 200 ml of a reaction solution with a composition, the same
as that described above except that the concentration of
1-deoxy-D-xylulose 5-phosphate was 0.15 mol/l, was incubated for 30
minutes at 37.degree. C. Then the whole amount of the reaction
solution was added to an activated carbon column, diluted with
water to 1 L, then added to a Dowex 1-X8 (C1-type, 3.5.times.20 cm)
column.
The solution was eluted with 400 ml of 1% saline solution, added to
a Sephadex G-10 (1.8.times.100 cm), then eluted with water. The
eluted fraction was freeze-dried, thereby isolating the reaction
product.
The molecular formula of the reaction product isolated from
HR-FABMS analysis was assumed to be C.sub.5H.sub.12O.sub.7 P [m/z
215.0276 (M--H), .DELTA.-4.5 mmu]. NMR analysis for .sup.1H and
.sup.13C resulted in the following chemical shifts.
.sup.1H NMR (D.sub.2O, 500 MHz): .delta. 4.03 (ddd, J=11.5, 6.5,
2.5 Hz, 1H), 3.84 (ddd, J=11.5, 8.0, 6.5 Hz, 1H), 3.78 (dd, J=80,
2.5 Hz, 1H), 3.60 (d, J=12.0 Hz, 1H), 3.50 (d, J=12.0 Hz,1H), 1.15
(s, 3 H); .sup.13C NMR (D.sub.2O, 125 MHz): .delta. 75.1 (C-2),
74.8 (C-3), 67.4 (C-1), 65.9 (C-4), 19.4 (2-Me)
The chemical shifts resulted from NMR analysis for .sup.1H and
.sup.13C of compounds obtained by treating the reaction products
with alkaline phosphatase (TAKARA SHUZO CO., LTD.) were completely
identical with that resulted from NMR analysis of
2-C-methyl-D-erythritol synthesized in the method described in
Tetrahedron Letter, 38, 6184 (1997).
Further the angle of rotation of the former compound was
[.alpha.].sub.D.sup.21=+6.0 (c=0.050, H.sub.2O), identical with the
angle of rotation [.alpha.].sub.D.sup.25=+7.0 (c=0.13, H.sub.2O) of
2-C-methyl-D-erythritol, reported in Tetrahedron Letter, 38, 6184
(1997).
These results reveal that the reaction product of yaeM gene product
was 2-C-methyl-D-erythritol 4-phosphate. That is, yaeM gene product
was found to have activity to yield 2-C-mehyl-D-erythritol
4-phosphate from 1deoxy-D-xylulose 5-phosphate with consumption of
NADPH. Based on this catalytic activity, this enzyme was named
1-deoxy-D-xylulose 5-phosphate reductoisomerase.
Characteristics of 1-deoxy-D-xylulose 5-Phosphate
Reductoisomerase
The enzymological characteristics of 1-deoxy-D-xylulose 5-phosphate
reductoisomerase were examined using 1 ml of the reaction system as
described in Example 8 (2). Here, 1 unit is defined as the activity
to oxidize 1 mmol of NADPH per a minute.
The activity decreased below 1/100 when NADPH was replaced with
NADH.
No reaction occurred when 1-deoxy-D-xylulose was used instead of
1-deoxy-D-xylulose 5-phosphate.
SDS-PAGE analysis showed that this enzyme was consisted of 42 kDa
polypeptide.
Table 9 shows effect on the reaction system by the addition of
metals.
TABLE-US-00009 TABLE 9 Effect of various metal ions on the activity
of 1-deoxy-D-xylulose 5-phosphate reductoisomerase Specific
Activity Additives (units/mg protein) none 0.3 EDTA N.D. MnCl.sub.2
11.8 CoCl.sub.2 6.0 MgCl.sub.2 4.0 CaCl.sub.2 0.2 NiSO.sub.4 0.2
ZnSO.sub.4 0.3 CuSO.sub.4 N.D. FeSO.sub.4 N.D.
These metal ions and EDTA were added such that the concentration of
each was 1 mol/l. N.D. indicates that no activity was detected.
Km for 1-deoxy-D-xylulose 5-phosphate and NADP in the presence of
MnCl.sub.2 were 249 .mu.mol/l and 7.4 .mu.mol/l, respectively.
FIG. 1 shows the effect of reaction temperature and FIG. 2 shows
the effect of reaction pH.
EXAMPLE 9
Construction and Characteristics of yaeM-Deleted Mutant
(1) Construction of yaeM-Disrupted Mutant
To test whether 1-deoxy-D-xylulose 5-phosphate reductoisomerase is
essential for cell growth or not, a 1-deoxy-D-xylulose 5-phosphate
reductoisomerase-deleted mutant was constructed as described
below.
A kanamycin-resistant gene cassette for insertion into yaeM gene
was produced as described below.
The plasmid pMEW41 obtained in Example 1 (2) was digested with a
restriction enzyme Bal I, and was subjected to agarose gel
electrophoresis, thereby obtaining a Bal I-treated DNA
fragment.
Tn5 was digested with restriction enzymes Hind III and Sam I, then
the both ends were blunt-ended using a DNA blunting kit (TAKARA
SHUZO CO., LTD.).
The resultant blunt-ended DNA fragments were mixed with previously
obtained Bal I-treated pMEW41 DNA fragments, and then the mixture
was treated with ethanol. Next the obtained DNA precipitate was
dissolved into 5 .mu.l of distilled water for ligation reaction to
occur, thereby obtaining recombinant DNA.
E. coli JM109 (purchased from TAKARA SHUZO CO., LTD.) was
transformed using this recombinant DNA according to standard
techniques. Next the transformant was spread on LB agar medium
containing 100 .mu.g/ml of ampicillin and 15 .mu.g/ml of kanamycin,
then cultured overnight at 37.degree. C.
Several ampicillin-resistant transformant colonies grown on the
medium were shake-cultured for 16 hours at 37.degree. C. in 10 ml
of LB liquid medium containing 100 .mu.g/ml of ampicillin and 15
.mu.g/ml of kanamycin.
The resulting culture was centrifuged to collect cells.
Plasmids were isolated from the cells according to the standard
techniques.
The plasmids isolated as described above were cleaved with various
restriction enzymes to test their structure. As a result, the
plasmid was confirmed to contain the DNA fragment of interest and
was named pMEW41Km.
yaeM gene on a chromosomal DNA of E. coli was disrupted by
homologous recombination using pMEW41Km. FIG. 3 shows the schematic
diagram for this recombination.
pMEW41Km was digested with restriction enzymes Hind III and Sac I,
subjected to agarose gel electrophoresis, thus purifying linear
fragments. E. coli FS1576 was transformed using the fragments
according to standard techniques. The strain FS1576 is available as
the strain ME9019 from National Institute of Genetics. The
transformants were spread on LB agar medium containing 15 .mu.g/ml
of kanamycin and 1 g/l of 2-C-methyl-D-erythritol, then cultured
overnight at 37.degree. C.
Several kanamycin-resistant colonies that grew on the medium were
shake-cultured for 16 hours at 37.degree. C. in 10 ml of LB liquid
medium containing 15 .mu.g/ml of kanamycin and 1 g/l of
2-C-methyl-D-erythritol.
The resulting culture was centrifuged to collect cells.
Chromosomal DNA was isolated from the cells by the standard
techniques.
The chromosomal DNA was digested with a restriction enzyme Sma I or
Pst I. Chromosomal DNA of the strain FS1576 was digested with a
restriction enzyme in the same way. These DNAs digested with
restriction enzymes were subjected to agarose gel electrophoresis
by the standard techniques, and then to Southern hybridization
analysis using the kanamycin-resistant gene and yaeM gene as
probes. Therefore, it was confirmed that the chromosomal DNA of the
kanamycin-resistant colonies had a structure as shown in FIG. 3,
that is, yaeM gene was disrupted by the kanamycin-resistant
gene.
(2) Characteristics of yaeM-Disrupted Mutant
The yaeM-disrupted strain produced as described above and its
parent strain FS1576 were spread on LB agar medium and the same
medium containing 1 g/l of 2-C-methyl-D-erythritol, then cultured
at 37.degree. C. Table 10 shows the cell growth after 2 days of
culture.
TABLE-US-00010 TABLE 10 Effect of deletion of yaeM gene on the E.
coli growth Cell growth on each medium Strain LB LB + ME*.sup.2
FS1576 + + yaeM-deleted strain - + *.sup.1Cell growth (+ indicates
good growth; - indicates no growth) *.sup.2ME indicates the
addition of 1 g/l of 2-C-methyl-D-erythritol.
No yaeM-deleted mutants grew on a medium lacking
2-C-methyl-D-erythritol. Therefore, This gene was shown to be
essential for the cell growth in the absence of
2-C-methyl-D-erythritol.
EXAMPLE 10
Effect of 1-Deoxy-D-xylulose 5-Phosphate Reductoisomerase Inhibitor
for Cell Growth
The following experiments were conducted based on the assumption
that fosmidomycin could inhibit 1-deoxy-D-xylulose 5-phosphate
reductoisomerase because 2-C-methyl-D-erythritol 4-phosphate, a
product from 1-deoxy-D-xylulose 5-phosphate reductoisomerase
reaction, or reaction intermediates expected to be produced in this
enzyme reaction is structurally analogous to fosmidomycin.
In the presence of fosmidomycin, the activity 1-deoxy-D-xylulose
5-phosphate reductoisomerase was measured by the method as
described in Example 8 in order to examine the effect on the
enzymatic activity.
Fosmidomycin had been synthesized according to the method described
in Chem. Pharm. Bull, 30, 111 118 (1982).
Total volume of reaction solution was reduced to 0.2 ml from the
volume of reaction solution described in Example 8 (2), but each
concentration was kept at the same level as the system of Example 8
. Fosmidomycin at various concentration was added to the reaction
solution, then the reaction was carried out at 37.degree. C. The
increase and decrease in NADPH were measured using Bench mark micro
plate reader (Bio-Rad Laboratories).
As shown in FIG. 4, fosmidomycin was shown to inhibit
1-deoxy-D-xylulose 5-phosphate reductoisomerase.
E. coli W3110 was spread on LB agar medium, the same medium
containing 3.13 mg/l of fosmidomycin, and the same medium
containing 3.13 mg/l of fosmidomycin and 0.25 g/l of
2-C-methyl-D-erythritol, then cultured at 37.degree. C.
Two days after culturing, the microorganism could grow on the two
types of media, that is, the LB agar medium and the same medium
containing fosmidomycin and 0.25 g/l of 2-C-methyl-D-erythritol,
but no microorganism grew on the LB agar medium supplemented only
with fosmidomycin.
These results clearly shows that fosmidomycin inhibited the cell
growth by inhibiting 1-deoxy-D-xylulose 5-phosphate
reductoisomerase. Accordingly, a substance inhibiting yaeM gene
product (1-deoxy-D-xylulose 5-phosphate reductoisomerase) activity
can be an effective antibiotic agent or herbicide.
All publications, patents and patent applications cited herein are
incorporated herein by reference in their entirety.
INDUSTRIAL APPLICABILITY
The present invention can provide a process for producing
isoprenoid compounds comprising integrating DNA into a vector
wherein the DNA contains one or more DNA involved in biosynthesis
of isoprenoid compounds useful in pharmaceuticals for cardiac
diseases, osteoporosis, homeostasis, prevention of cancer, and
immunopotentiation, health food and anti-fouling paint products
against barnacles, introducing the resultant recombinant DNA into a
host cell derived from prokaryote, culturing the obtained
transformants in a medium, allowing the transformant to produce and
accumulate isoprenoid compounds in the culture, and recovering the
isoprenoid compounds from the culture, a process for producing a
protein having activity to improve efficiency in the biosynthesis
of isoprenoid compounds comprising integrating DNA containing one
or more DNA encoding the protein into a vector, introducing the
resultant recombinant DNA into a host cell, culturing the obtained
transformant in a medium, allowing the transformant to produce and
accumulate said protein in the culture, and recovering said protein
from the culture; the protein; and novel enzymatic protein having
activity to catalyze a reaction to produce 2-C-methyl-D-erythritol
4-phosphate from 1-deoxy-D-xylulose 5-phosphate; and a method for
screening a compound with antibiotic and/or weeding activity
comprising screening a substance inhibiting the enzyme.
SEQUENCE LISTING FREE TEXT
SEQ ID NO:12: synthetic DNA SEQ ID NO:13: synthetic DNA SEQ ID
NO:14: synthetic DNA SEQ ID NO:15: synthetic DNA SEQ ID NO:16:
synthetic DNA SEQ ID NO:17: synthetic DNA SEQ ID NO:18: synthetic
DNA SEQ ID NO:19: synthetic DNA SEQ ID NO:20: synthetic DNA SEQ ID
NO:21: synthetic DNA SEQ ID NO:22: synthetic DNA SEQ ID NO:23:
synthetic DNA SEQ ID NO:24: synthetic DNA SEQ ID NO:25: synthetic
DNA SEQ ID NO:32: synthetic DNA SEQ ID NO:33: synthetic DNA SEQ ID
NO:34: synthetic DNA
SEQUENCE LISTINGS
1
34 1 620 PRT Escherichia coli 1 Met Ser Phe Asp Ile Ala Lys Tyr Pro
Thr Leu Ala Leu Val Asp Ser 1 5 10 15 Thr Gln Glu Leu Arg Leu Leu
Pro Lys Glu Ser Leu Pro Lys Leu Cys 20 25 30 Asp Glu Leu Arg Arg
Tyr Leu Leu Asp Ser Val Ser Arg Ser Ser Gly 35 40 45 His Phe Ala
Ser Gly Leu Gly Thr Val Glu Leu Thr Val Ala Leu His 50 55 60 Tyr
Val Tyr Asn Thr Pro Phe Asp Gln Leu Ile Trp Asp Val Gly His 65 70
75 80 Gln Ala Tyr Pro His Lys Ile Leu Thr Gly Arg Arg Asp Lys Ile
Gly 85 90 95 Thr Ile Arg Gln Lys Gly Gly Leu His Pro Phe Pro Trp
Arg Gly Glu 100 105 110 Ser Glu Tyr Asp Val Leu Ser Val Gly His Ser
Ser Thr Ser Ile Ser 115 120 125 Ala Gly Ile Gly Ile Ala Val Ala Ala
Glu Lys Glu Gly Lys Asn Arg 130 135 140 Arg Thr Val Cys Val Ile Gly
Asp Gly Ala Ile Thr Ala Gly Met Ala 145 150 155 160 Phe Glu Ala Met
Asn His Ala Gly Asp Ile Arg Pro Asp Met Leu Val 165 170 175 Ile Leu
Asn Asp Asn Glu Met Ser Ile Ser Glu Asn Val Gly Ala Leu 180 185 190
Asn Asn His Leu Ala Gln Leu Leu Ser Gly Lys Leu Tyr Ser Ser Leu 195
200 205 Arg Glu Gly Gly Lys Lys Val Phe Ser Gly Val Pro Pro Ile Lys
Glu 210 215 220 Leu Leu Lys Arg Thr Glu Glu His Ile Lys Gly Met Val
Val Pro Gly 225 230 235 240 Thr Leu Phe Glu Glu Leu Gly Phe Asn Tyr
Ile Gly Pro Val Asp Gly 245 250 255 His Asp Val Leu Gly Leu Ile Thr
Thr Leu Lys Asn Met Arg Asp Leu 260 265 270 Lys Gly Pro Gln Phe Leu
His Ile Met Thr Lys Lys Gly Arg Gly Tyr 275 280 285 Glu Pro Ala Glu
Lys Asp Pro Ile Thr Phe His Ala Val Pro Lys Phe 290 295 300 Asp Pro
Ser Ser Gly Cys Leu Pro Lys Ser Ser Gly Gly Leu Pro Ser 305 310 315
320 Tyr Ser Lys Ile Phe Gly Asp Trp Leu Cys Glu Thr Ala Ala Lys Asp
325 330 335 Asn Lys Leu Met Ala Ile Thr Pro Ala Met Arg Glu Gly Ser
Gly Met 340 345 350 Val Glu Phe Ser Arg Lys Phe Pro Asp Arg Tyr Phe
Asp Val Ala Ile 355 360 365 Ala Glu Gln His Ala Val Thr Phe Ala Ala
Gly Leu Ala Ile Gly Gly 370 375 380 Tyr Lys Pro Ile Val Ala Ile Tyr
Ser Thr Phe Leu Gln Arg Ala Tyr 385 390 395 400 Asp Gln Val Leu His
Asp Val Ala Ile Gln Lys Leu Pro Val Leu Phe 405 410 415 Ala Ile Asp
Arg Ala Gly Ile Val Gly Ala Asp Gly Gln Thr His Gln 420 425 430 Gly
Ala Phe Asp Leu Ser Tyr Leu Arg Cys Ile Pro Glu Met Val Ile 435 440
445 Met Thr Pro Ser Asp Glu Asn Glu Cys Arg Gln Met Leu Tyr Thr Gly
450 455 460 Tyr His Tyr Asn Asp Gly Pro Ser Ala Val Arg Tyr Pro Arg
Gly Asn 465 470 475 480 Ala Val Gly Val Glu Leu Thr Pro Leu Glu Lys
Leu Pro Ile Gly Lys 485 490 495 Gly Ile Val Lys Arg Arg Gly Glu Lys
Leu Ala Ile Leu Asn Phe Gly 500 505 510 Thr Leu Met Pro Glu Ala Ala
Lys Val Ala Glu Ser Leu Asn Ala Thr 515 520 525 Leu Val Asp Met Arg
Phe Val Lys Pro Leu Asp Glu Ala Leu Ile Leu 530 535 540 Glu Met Ala
Ala Ser His Glu Ala Leu Val Thr Val Glu Glu Asn Ala 545 550 555 560
Ile Met Gly Gly Ala Gly Ser Gly Val Asn Glu Val Leu Met Ala His 565
570 575 Arg Lys Pro Val Pro Val Leu Asn Ile Gly Leu Pro Asp Phe Phe
Ile 580 585 590 Pro Gln Gly Thr Gln Glu Glu Met Arg Ala Glu Leu Gly
Leu Asp Ala 595 600 605 Ala Gly Met Glu Ala Lys Ile Lys Ala Trp Leu
Ala 610 615 620 2 299 PRT Escherichia coli 2 Met Asp Phe Pro Gln
Gln Leu Glu Ala Cys Val Lys Gln Ala Asn Gln 1 5 10 15 Ala Leu Ser
Arg Phe Ile Ala Pro Leu Pro Phe Gln Asn Thr Pro Val 20 25 30 Val
Glu Thr Met Gln Tyr Gly Ala Leu Leu Gly Gly Lys Arg Leu Arg 35 40
45 Pro Phe Leu Val Tyr Ala Thr Gly His Met Phe Gly Val Ser Thr Asn
50 55 60 Thr Leu Asp Ala Pro Ala Ala Ala Val Glu Cys Ile His Ala
Tyr Ser 65 70 75 80 Leu Ile His Asp Asp Leu Pro Ala Met Asp Asp Asp
Asp Leu Arg Arg 85 90 95 Gly Leu Pro Thr Cys His Val Lys Phe Gly
Glu Ala Asn Ala Ile Leu 100 105 110 Ala Gly Asp Ala Leu Gln Thr Leu
Ala Phe Ser Ile Leu Ser Asp Ala 115 120 125 Asp Met Pro Glu Val Ser
Asp Arg Asp Arg Ile Ser Met Ile Ser Glu 130 135 140 Leu Ala Ser Ala
Ser Gly Ile Ala Gly Met Cys Gly Gly Gln Ala Leu 145 150 155 160 Asp
Leu Asp Ala Glu Gly Lys His Val Pro Leu Asp Ala Leu Glu Arg 165 170
175 Ile His Arg His Lys Thr Gly Ala Leu Ile Arg Ala Ala Val Arg Leu
180 185 190 Gly Ala Leu Ser Ala Gly Asp Lys Gly Arg Arg Ala Leu Pro
Val Leu 195 200 205 Asp Lys Tyr Ala Glu Ser Ile Gly Leu Ala Phe Gln
Val Gln Asp Asp 210 215 220 Ile Leu Asp Val Val Gly Asp Thr Ala Thr
Leu Gly Lys Arg Gln Gly 225 230 235 240 Ala Asp Gln Gln Leu Gly Lys
Ser Thr Tyr Pro Ala Leu Leu Gly Leu 245 250 255 Glu Gln Ala Arg Lys
Lys Ala Arg Asp Leu Ile Asp Asp Ala Arg Gln 260 265 270 Ser Leu Lys
Gln Leu Ala Glu Gln Ser Leu Asp Thr Ser Ala Leu Glu 275 280 285 Ala
Leu Ala Asp Tyr Ile Ile Gln Arg Asn Lys 290 295 3 80 PRT
Escherichia coli 3 Met Pro Lys Lys Asn Glu Ala Pro Ala Ser Phe Glu
Lys Ala Leu Ser 1 5 10 15 Glu Leu Glu Gln Ile Val Thr Arg Leu Glu
Ser Gly Asp Leu Pro Leu 20 25 30 Glu Glu Ala Leu Asn Glu Phe Glu
Arg Gly Val Gln Leu Ala Arg Gln 35 40 45 Gly Gln Ala Lys Leu Gln
Gln Ala Glu Gln Arg Val Gln Ile Leu Leu 50 55 60 Ser Asp Asn Glu
Asp Ala Ser Leu Thr Pro Phe Thr Pro Asp Asn Glu 65 70 75 80 4 348
PRT Escherichia coli 4 Val Thr Gly Val Asn Glu Cys Ser Arg Ser Thr
Cys Asn Leu Lys Tyr 1 5 10 15 Asp Glu Tyr Ser Arg Ser Gly Ser Met
Gln Tyr Asn Pro Leu Gly Lys 20 25 30 Thr Asp Leu Arg Val Ser Arg
Leu Cys Leu Gly Cys Met Thr Phe Gly 35 40 45 Glu Pro Asp Arg Gly
Asn His Ala Trp Thr Leu Pro Glu Glu Ser Ser 50 55 60 Arg Pro Ile
Ile Lys Arg Ala Leu Glu Gly Gly Ile Asn Phe Phe Asp 65 70 75 80 Thr
Ala Asn Ser Tyr Ser Asp Gly Ser Ser Glu Glu Ile Val Gly Arg 85 90
95 Ala Leu Arg Asp Phe Ala Arg Arg Glu Asp Val Val Val Ala Thr Lys
100 105 110 Val Phe His Arg Val Gly Asp Leu Pro Glu Gly Leu Ser Arg
Ala Gln 115 120 125 Ile Leu Arg Ser Ile Asp Asp Ser Leu Arg Arg Leu
Gly Met Asp Tyr 130 135 140 Val Asp Ile Leu Gln Ile His Arg Trp Asp
Tyr Asn Thr Pro Ile Glu 145 150 155 160 Glu Thr Leu Glu Ala Leu Asn
Asp Val Val Lys Ala Gly Lys Ala Arg 165 170 175 Tyr Ile Gly Ala Ser
Ser Met His Ala Ser Gln Phe Ala Gln Ala Leu 180 185 190 Glu Leu Gln
Lys Gln His Gly Trp Ala Gln Phe Val Ser Met Gln Asp 195 200 205 His
Tyr Asn Leu Ile Tyr Arg Glu Glu Glu Arg Glu Met Leu Pro Leu 210 215
220 Cys Tyr Gln Glu Gly Val Ala Val Ile Pro Trp Ser Pro Leu Ala Arg
225 230 235 240 Gly Arg Leu Thr Arg Pro Trp Gly Glu Thr Thr Ala Arg
Leu Val Ser 245 250 255 Asp Glu Val Gly Lys Asn Leu Tyr Lys Glu Ser
Asp Glu Asn Asp Ala 260 265 270 Gln Ile Ala Glu Arg Leu Thr Gly Val
Ser Glu Glu Leu Gly Ala Thr 275 280 285 Arg Ala Gln Val Ala Leu Ala
Trp Leu Leu Ser Lys Pro Gly Ile Ala 290 295 300 Ala Pro Ile Ile Gly
Thr Ser Arg Glu Glu Gln Leu Asp Glu Leu Leu 305 310 315 320 Asn Ala
Val Asp Ile Thr Leu Lys Pro Glu Gln Ile Ala Glu Leu Glu 325 330 335
Thr Pro Tyr Lys Pro His Pro Val Val Gly Phe Lys 340 345 5 398 PRT
Escherichia coli 5 Met Lys Gln Leu Thr Ile Leu Gly Ser Thr Gly Ser
Ile Gly Cys Ser 1 5 10 15 Thr Leu Asp Val Val Arg His Asn Pro Glu
His Phe Arg Val Val Ala 20 25 30 Leu Val Ala Gly Lys Asn Val Thr
Arg Met Val Glu Gln Cys Leu Glu 35 40 45 Phe Ser Pro Arg Tyr Ala
Val Met Asp Asp Glu Ala Ser Ala Lys Leu 50 55 60 Leu Lys Thr Met
Leu Gln Gln Gln Gly Ser Arg Thr Glu Val Leu Ser 65 70 75 80 Gly Gln
Gln Ala Ala Cys Asp Met Ala Ala Leu Glu Asp Val Asp Gln 85 90 95
Val Met Ala Ala Ile Val Gly Ala Ala Gly Leu Leu Pro Thr Leu Ala 100
105 110 Ala Ile Arg Ala Gly Lys Thr Ile Leu Leu Ala Asn Lys Glu Ser
Leu 115 120 125 Val Thr Cys Gly Arg Leu Phe Met Asp Ala Val Lys Gln
Ser Lys Ala 130 135 140 Gln Leu Leu Pro Val Asp Ser Glu His Asn Ala
Ile Phe Gln Ser Leu 145 150 155 160 Pro Gln Pro Ile Gln His Asn Leu
Gly Tyr Ala Asp Leu Glu Gln Asn 165 170 175 Gly Val Val Ser Ile Leu
Leu Thr Gly Ser Gly Gly Pro Phe Arg Glu 180 185 190 Thr Pro Leu Arg
Asp Leu Ala Thr Met Thr Pro Asp Gln Ala Cys Arg 195 200 205 His Pro
Asn Trp Ser Met Gly Arg Lys Ile Ser Val Asp Ser Ala Thr 210 215 220
Met Met Asn Lys Gly Leu Glu Tyr Ile Glu Ala Arg Trp Leu Phe Asn 225
230 235 240 Ala Ser Ala Ser Gln Met Glu Val Leu Ile His Pro Gln Ser
Val Ile 245 250 255 His Ser Met Val Arg Tyr Gln Asp Gly Ser Val Leu
Ala Gln Leu Gly 260 265 270 Glu Pro Asp Met Val Arg Gln Leu Pro Thr
Pro Trp Ala Trp Pro Asn 275 280 285 Arg Val Asn Ser Gly Val Lys Pro
Leu Asp Phe Cys Lys Leu Ser Ala 290 295 300 Leu Thr Phe Ala Ala Pro
Asp Tyr Asp Arg Tyr Pro Cys Leu Lys Leu 305 310 315 320 Ala Met Glu
Ala Phe Glu Gln Gly Gln Ala Ala Thr Thr Ala Leu Asn 325 330 335 Ala
Ala Asn Glu Ile Thr Val Ala Ala Phe Leu Ala Gln Gln Ile Arg 340 345
350 Phe Thr Asp Ile Ala Ala Leu Asn Leu Ser Val Leu Glu Lys Met Asp
355 360 365 Met Arg Glu Pro Gln Cys Val Asp Asp Val Leu Ser Val Asp
Ala Asn 370 375 380 Ala Arg Glu Val Ala Arg Lys Glu Val Met Arg Leu
Ala Ser 385 390 395 6 1860 DNA Escherichia coli CDS (1)..(1860) 6
atg agt ttt gat att gcc aaa tac ccg acc ctg gca ctg gtc gac tcc 48
Met Ser Phe Asp Ile Ala Lys Tyr Pro Thr Leu Ala Leu Val Asp Ser 1 5
10 15 acc cag gag tta cga ctg ttg ccg aaa gag agt tta ccg aaa ctc
tgc 96 Thr Gln Glu Leu Arg Leu Leu Pro Lys Glu Ser Leu Pro Lys Leu
Cys 20 25 30 gac gaa ctg cgc cgc tat tta ctc gac agc gtg agc cgt
tcc agc ggg 144 Asp Glu Leu Arg Arg Tyr Leu Leu Asp Ser Val Ser Arg
Ser Ser Gly 35 40 45 cac ttc gcc tcc ggg ctg ggc acg gtc gaa ctg
acc gtg gcg ctg cac 192 His Phe Ala Ser Gly Leu Gly Thr Val Glu Leu
Thr Val Ala Leu His 50 55 60 tat gtc tac aac acc ccg ttt gac caa
ttg att tgg gat gtg ggg cat 240 Tyr Val Tyr Asn Thr Pro Phe Asp Gln
Leu Ile Trp Asp Val Gly His 65 70 75 80 cag gct tat ccg cat aaa att
ttg acc gga cgc cgc gac aaa atc ggc 288 Gln Ala Tyr Pro His Lys Ile
Leu Thr Gly Arg Arg Asp Lys Ile Gly 85 90 95 acc atc cgt cag aaa
ggc ggt ctg cac ccg ttc ccg tgg cgc ggc gaa 336 Thr Ile Arg Gln Lys
Gly Gly Leu His Pro Phe Pro Trp Arg Gly Glu 100 105 110 agc gaa tat
gac gta tta agc gtc ggg cat tca tca acc tcc atc agt 384 Ser Glu Tyr
Asp Val Leu Ser Val Gly His Ser Ser Thr Ser Ile Ser 115 120 125 gcc
gga att ggt att gcg gtt gct gcc gaa aaa gaa ggc aaa aat cgc 432 Ala
Gly Ile Gly Ile Ala Val Ala Ala Glu Lys Glu Gly Lys Asn Arg 130 135
140 cgc acc gtc tgt gtc att ggc gat ggc gcg att acc gca ggc atg gcg
480 Arg Thr Val Cys Val Ile Gly Asp Gly Ala Ile Thr Ala Gly Met Ala
145 150 155 160 ttt gaa gcg atg aat cac gcg ggc gat atc cgt cct gat
atg ctg gtg 528 Phe Glu Ala Met Asn His Ala Gly Asp Ile Arg Pro Asp
Met Leu Val 165 170 175 att ctc aac gac aat gaa atg tcg att tcc gaa
aat gtc ggc gcg ctc 576 Ile Leu Asn Asp Asn Glu Met Ser Ile Ser Glu
Asn Val Gly Ala Leu 180 185 190 aac aac cat ctg gca cag ctg ctt tcc
ggt aag ctt tac tct tca ctg 624 Asn Asn His Leu Ala Gln Leu Leu Ser
Gly Lys Leu Tyr Ser Ser Leu 195 200 205 cgc gaa ggc ggg aaa aaa gtt
ttc tct ggc gtg ccg cca att aaa gag 672 Arg Glu Gly Gly Lys Lys Val
Phe Ser Gly Val Pro Pro Ile Lys Glu 210 215 220 ctg ctc aaa cgc acc
gaa gaa cat att aaa ggc atg gta gtg cct ggc 720 Leu Leu Lys Arg Thr
Glu Glu His Ile Lys Gly Met Val Val Pro Gly 225 230 235 240 acg ttg
ttt gaa gag ctg ggc ttt aac tac atc ggc ccg gtg gac ggt 768 Thr Leu
Phe Glu Glu Leu Gly Phe Asn Tyr Ile Gly Pro Val Asp Gly 245 250 255
cac gat gtg ctg ggg ctt atc acc acg cta aag aac atg cgc gac ctg 816
His Asp Val Leu Gly Leu Ile Thr Thr Leu Lys Asn Met Arg Asp Leu 260
265 270 aaa ggc ccg cag ttc ctg cat atc atg acc aaa aaa ggt cgt ggt
tat 864 Lys Gly Pro Gln Phe Leu His Ile Met Thr Lys Lys Gly Arg Gly
Tyr 275 280 285 gaa ccg gca gaa aaa gac ccg atc act ttc cac gcc gtg
cct aaa ttt 912 Glu Pro Ala Glu Lys Asp Pro Ile Thr Phe His Ala Val
Pro Lys Phe 290 295 300 gat ccc tcc agc ggt tgt ttg ccg aaa agt agc
ggc ggt ttg ccg agc 960 Asp Pro Ser Ser Gly Cys Leu Pro Lys Ser Ser
Gly Gly Leu Pro Ser 305 310 315 320 tat tca aaa atc ttt ggc gac tgg
ttg tgc gaa acg gca gcg aaa gac 1008 Tyr Ser Lys Ile Phe Gly Asp
Trp Leu Cys Glu Thr Ala Ala Lys Asp 325 330 335 aac aag ctg atg gcg
att act ccg gcg atg cgt gaa ggt tcc ggc atg 1056 Asn Lys Leu Met
Ala Ile Thr Pro Ala Met Arg Glu Gly Ser Gly Met 340 345 350 gtc gag
ttt tca cgt aaa ttc ccg gat cgc tac ttc gac gtg gca att 1104 Val
Glu Phe Ser Arg Lys Phe Pro Asp Arg Tyr Phe Asp Val Ala Ile 355 360
365 gcc gag caa cac gcg gtg acc ttt gct gcg ggt ctg gcg att ggt ggg
1152 Ala Glu Gln His Ala Val Thr Phe Ala Ala Gly Leu Ala Ile Gly
Gly 370 375 380 tac aaa ccc att gtc gcg att tac tcc act ttc ctg caa
cgc gcc tat 1200 Tyr Lys Pro Ile Val Ala Ile Tyr Ser Thr Phe Leu
Gln Arg Ala Tyr 385 390 395 400 gat cag gtg ctg cat gac gtg gcg att
caa aag ctt ccg gtc ctg ttc 1248 Asp Gln Val Leu His Asp Val Ala
Ile Gln Lys Leu Pro Val Leu Phe 405 410 415 gcc atc gac cgc gcg ggc
att gtt ggt gct gac ggt caa acc cat cag 1296 Ala
Ile Asp Arg Ala Gly Ile Val Gly Ala Asp Gly Gln Thr His Gln 420 425
430 ggt gct ttt gat ctc tct tac ctg cgc tgc ata ccg gaa atg gtc att
1344 Gly Ala Phe Asp Leu Ser Tyr Leu Arg Cys Ile Pro Glu Met Val
Ile 435 440 445 atg acc ccg agc gat gaa aac gaa tgt cgc cag atg ctc
tat acc ggc 1392 Met Thr Pro Ser Asp Glu Asn Glu Cys Arg Gln Met
Leu Tyr Thr Gly 450 455 460 tat cac tat aac gat ggc ccg tca gcg gtg
cgc tac ccg cgt ggc aac 1440 Tyr His Tyr Asn Asp Gly Pro Ser Ala
Val Arg Tyr Pro Arg Gly Asn 465 470 475 480 gcg gtc ggc gtg gaa ctg
acg ccg ctg gaa aaa cta cca att ggc aaa 1488 Ala Val Gly Val Glu
Leu Thr Pro Leu Glu Lys Leu Pro Ile Gly Lys 485 490 495 ggc att gtg
aag cgt cgt ggc gag aaa ctg gcg atc ctt aac ttt ggt 1536 Gly Ile
Val Lys Arg Arg Gly Glu Lys Leu Ala Ile Leu Asn Phe Gly 500 505 510
acg ctg atg cca gaa gcg gcg aaa gtc gcc gaa tcg ctg aac gcc acg
1584 Thr Leu Met Pro Glu Ala Ala Lys Val Ala Glu Ser Leu Asn Ala
Thr 515 520 525 ctg gtc gat atg cgt ttt gtg aaa ccg ctt gat gaa gcg
tta att ctg 1632 Leu Val Asp Met Arg Phe Val Lys Pro Leu Asp Glu
Ala Leu Ile Leu 530 535 540 gaa atg gcc gcc agc cat gaa gcg ctg gtc
acc gta gaa gaa aac gcc 1680 Glu Met Ala Ala Ser His Glu Ala Leu
Val Thr Val Glu Glu Asn Ala 545 550 555 560 att atg ggc ggc gca ggc
agc ggc gtg aac gaa gtg ctg atg gcc cat 1728 Ile Met Gly Gly Ala
Gly Ser Gly Val Asn Glu Val Leu Met Ala His 565 570 575 cgt aaa cca
gta ccc gtg ctg aac att ggc ctg ccg gac ttc ttt att 1776 Arg Lys
Pro Val Pro Val Leu Asn Ile Gly Leu Pro Asp Phe Phe Ile 580 585 590
ccg caa gga act cag gaa gaa atg cgc gcc gaa ctc ggc ctc gat gcc
1824 Pro Gln Gly Thr Gln Glu Glu Met Arg Ala Glu Leu Gly Leu Asp
Ala 595 600 605 gct ggt atg gaa gcc aaa atc aag gcc tgg ctg gca
1860 Ala Gly Met Glu Ala Lys Ile Lys Ala Trp Leu Ala 610 615 620 7
897 DNA Escherichia coli CDS (1)..(897) 7 atg gac ttt ccg cag caa
ctc gaa gcc tgc gtt aag cag gcc aac cag 48 Met Asp Phe Pro Gln Gln
Leu Glu Ala Cys Val Lys Gln Ala Asn Gln 1 5 10 15 gcg ctg agc cgt
ttt atc gcc cca ctg ccc ttt cag aac act ccc gtg 96 Ala Leu Ser Arg
Phe Ile Ala Pro Leu Pro Phe Gln Asn Thr Pro Val 20 25 30 gtc gaa
acc atg cag tat ggc gca tta tta ggt ggt aag cgc ctg cga 144 Val Glu
Thr Met Gln Tyr Gly Ala Leu Leu Gly Gly Lys Arg Leu Arg 35 40 45
cct ttc ctg gtt tat gcc acc ggt cat atg ttc ggc gtt agc aca aac 192
Pro Phe Leu Val Tyr Ala Thr Gly His Met Phe Gly Val Ser Thr Asn 50
55 60 acg ctg gac gca ccc gct gcc gcc gtt gag tgt atc cac gct tac
tca 240 Thr Leu Asp Ala Pro Ala Ala Ala Val Glu Cys Ile His Ala Tyr
Ser 65 70 75 80 tta att cat gat gat tta ccg gca atg gat gat gac gat
ctg cgt cgc 288 Leu Ile His Asp Asp Leu Pro Ala Met Asp Asp Asp Asp
Leu Arg Arg 85 90 95 ggt ttg cca acc tgc cat gtg aag ttt ggc gaa
gca aac gcg att ctc 336 Gly Leu Pro Thr Cys His Val Lys Phe Gly Glu
Ala Asn Ala Ile Leu 100 105 110 gct ggc gac gct tta caa acg ctg gcg
ttc tcg att tta agc gat gcc 384 Ala Gly Asp Ala Leu Gln Thr Leu Ala
Phe Ser Ile Leu Ser Asp Ala 115 120 125 gat atg ccg gaa gtg tcg gac
cgc gac aga att tcg atg att tct gaa 432 Asp Met Pro Glu Val Ser Asp
Arg Asp Arg Ile Ser Met Ile Ser Glu 130 135 140 ctg gcg agc gcc agt
ggt att gcc gga atg tgc ggt ggt cag gca tta 480 Leu Ala Ser Ala Ser
Gly Ile Ala Gly Met Cys Gly Gly Gln Ala Leu 145 150 155 160 gat tta
gac gcg gaa ggc aaa cac gta cct ctg gac gcg ctt gag cgt 528 Asp Leu
Asp Ala Glu Gly Lys His Val Pro Leu Asp Ala Leu Glu Arg 165 170 175
att cat cgt cat aaa acc ggc gca ttg att cgc gcc gcc gtt cgc ctt 576
Ile His Arg His Lys Thr Gly Ala Leu Ile Arg Ala Ala Val Arg Leu 180
185 190 ggt gca tta agc gcc gga gat aaa gga cgt cgt gct ctg ccg gta
ctc 624 Gly Ala Leu Ser Ala Gly Asp Lys Gly Arg Arg Ala Leu Pro Val
Leu 195 200 205 gac aag tat gca gag agc atc ggc ctt gcc ttc cag gtt
cag gat gac 672 Asp Lys Tyr Ala Glu Ser Ile Gly Leu Ala Phe Gln Val
Gln Asp Asp 210 215 220 atc ctg gat gtg gtg gga gat act gca acg ttg
gga aaa cgc cag ggt 720 Ile Leu Asp Val Val Gly Asp Thr Ala Thr Leu
Gly Lys Arg Gln Gly 225 230 235 240 gcc gac cag caa ctt ggt aaa agt
acc tac cct gca ctt ctg ggt ctt 768 Ala Asp Gln Gln Leu Gly Lys Ser
Thr Tyr Pro Ala Leu Leu Gly Leu 245 250 255 gag caa gcc cgg aag aaa
gcc cgg gat ctg atc gac gat gcc cgt cag 816 Glu Gln Ala Arg Lys Lys
Ala Arg Asp Leu Ile Asp Asp Ala Arg Gln 260 265 270 tcg ctg aaa caa
ctg gct gaa cag tca ctc gat acc tcg gca ctg gaa 864 Ser Leu Lys Gln
Leu Ala Glu Gln Ser Leu Asp Thr Ser Ala Leu Glu 275 280 285 gcg cta
gcg gac tac atc atc cag cgt aat aaa 897 Ala Leu Ala Asp Tyr Ile Ile
Gln Arg Asn Lys 290 295 8 240 DNA Escherichia coli CDS (1)..(240) 8
atg ccg aag aaa aat gag gcg ccc gcc agc ttt gaa aag gcg ctg agc 48
Met Pro Lys Lys Asn Glu Ala Pro Ala Ser Phe Glu Lys Ala Leu Ser 1 5
10 15 gag ctg gaa cag att gta acc cgt ctg gaa agt ggc gac ctg ccg
ctg 96 Glu Leu Glu Gln Ile Val Thr Arg Leu Glu Ser Gly Asp Leu Pro
Leu 20 25 30 gaa gag gcg ctg aac gag ttc gaa cgc ggc gtg cag ctg
gca cgt cag 144 Glu Glu Ala Leu Asn Glu Phe Glu Arg Gly Val Gln Leu
Ala Arg Gln 35 40 45 ggg cag gcc aaa tta caa caa gcc gaa cag cgc
gta caa att ctg ctg 192 Gly Gln Ala Lys Leu Gln Gln Ala Glu Gln Arg
Val Gln Ile Leu Leu 50 55 60 tct gac aat gaa gac gcc tct cta acc
cct ttt aca ccg gac aat gag 240 Ser Asp Asn Glu Asp Ala Ser Leu Thr
Pro Phe Thr Pro Asp Asn Glu 65 70 75 80 9 1044 DNA Escherichia coli
CDS (1)..(1044) 9 gtg act ggg gtg aac gaa tgc agc cgc agc aca tgc
aac ttg aag tat 48 Val Thr Gly Val Asn Glu Cys Ser Arg Ser Thr Cys
Asn Leu Lys Tyr 1 5 10 15 gac gag tat agc agg agt ggc agc atg caa
tac aac ccc tta gga aaa 96 Asp Glu Tyr Ser Arg Ser Gly Ser Met Gln
Tyr Asn Pro Leu Gly Lys 20 25 30 acc gac ctt cgc gtt tcc cga ctt
tgc ctc ggc tgt atg acc ttt ggc 144 Thr Asp Leu Arg Val Ser Arg Leu
Cys Leu Gly Cys Met Thr Phe Gly 35 40 45 gag cca gat cgc ggt aat
cac gca tgg aca ctg ccg gaa gaa agc agc 192 Glu Pro Asp Arg Gly Asn
His Ala Trp Thr Leu Pro Glu Glu Ser Ser 50 55 60 cgt ccc ata att
aaa cgt gca ctg gaa ggc ggc ata aat ttc ttt gat 240 Arg Pro Ile Ile
Lys Arg Ala Leu Glu Gly Gly Ile Asn Phe Phe Asp 65 70 75 80 acc gcc
aac agt tat tct gac ggc agc agc gaa gag atc gtc ggt cgc 288 Thr Ala
Asn Ser Tyr Ser Asp Gly Ser Ser Glu Glu Ile Val Gly Arg 85 90 95
gca ctg cgg gat ttc gcc cgt cgt gaa gac gtg gtc gtt gcg acc aaa 336
Ala Leu Arg Asp Phe Ala Arg Arg Glu Asp Val Val Val Ala Thr Lys 100
105 110 gtg ttc cat cgc gtt ggt gat tta ccg gaa gga tta tcc cgt gcg
caa 384 Val Phe His Arg Val Gly Asp Leu Pro Glu Gly Leu Ser Arg Ala
Gln 115 120 125 att ttg cgc tct atc gac gac agc ctg cga cgt ctc ggc
atg gat tat 432 Ile Leu Arg Ser Ile Asp Asp Ser Leu Arg Arg Leu Gly
Met Asp Tyr 130 135 140 gtc gat atc ctg caa att cat cgc tgg gat tac
aac acg ccg atc gaa 480 Val Asp Ile Leu Gln Ile His Arg Trp Asp Tyr
Asn Thr Pro Ile Glu 145 150 155 160 gag acg ctg gaa gcc ctc aac gac
gtg gta aaa gcc ggg aaa gcg cgt 528 Glu Thr Leu Glu Ala Leu Asn Asp
Val Val Lys Ala Gly Lys Ala Arg 165 170 175 tat atc ggc gcg tca tca
atg cac gct tcg cag ttt gct cag gca ctg 576 Tyr Ile Gly Ala Ser Ser
Met His Ala Ser Gln Phe Ala Gln Ala Leu 180 185 190 gaa ctc caa aaa
cag cac ggc tgg gcg cag ttt gtc agt atg cag gat 624 Glu Leu Gln Lys
Gln His Gly Trp Ala Gln Phe Val Ser Met Gln Asp 195 200 205 cac tac
aat ctg att tat cgt gaa gaa gag cgc gag atg cta cca ctg 672 His Tyr
Asn Leu Ile Tyr Arg Glu Glu Glu Arg Glu Met Leu Pro Leu 210 215 220
tgt tat cag gag ggc gtg gcg gta att cca tgg agc ccg ctg gca agg 720
Cys Tyr Gln Glu Gly Val Ala Val Ile Pro Trp Ser Pro Leu Ala Arg 225
230 235 240 ggc cgt ctg acg cgt ccg tgg gga gaa act acc gca cga ctg
gtg tct 768 Gly Arg Leu Thr Arg Pro Trp Gly Glu Thr Thr Ala Arg Leu
Val Ser 245 250 255 gat gag gtg ggg aaa aat ctc tat aaa gaa agc gat
gaa aat gac gcg 816 Asp Glu Val Gly Lys Asn Leu Tyr Lys Glu Ser Asp
Glu Asn Asp Ala 260 265 270 cag atc gca gag cgg tta aca ggc gtc agt
gaa gaa ctg ggg gcg aca 864 Gln Ile Ala Glu Arg Leu Thr Gly Val Ser
Glu Glu Leu Gly Ala Thr 275 280 285 cga gca caa gtt gcg ctg gcc tgg
ttg ttg agt aaa ccg ggc att gcc 912 Arg Ala Gln Val Ala Leu Ala Trp
Leu Leu Ser Lys Pro Gly Ile Ala 290 295 300 gca ccg att atc gga act
tcg cgc gaa gaa cag ctt gat gag cta ttg 960 Ala Pro Ile Ile Gly Thr
Ser Arg Glu Glu Gln Leu Asp Glu Leu Leu 305 310 315 320 aac gcg gtg
gat atc act ttg aag ccg gaa cag att gcc gaa ctg gaa 1008 Asn Ala
Val Asp Ile Thr Leu Lys Pro Glu Gln Ile Ala Glu Leu Glu 325 330 335
acg ccg tat aaa ccg cat cct gtc gta gga ttt aaa 1044 Thr Pro Tyr
Lys Pro His Pro Val Val Gly Phe Lys 340 345 10 1194 DNA Escherichia
coli CDS (1)..(1194) 10 atg aag caa ctc acc att ctg ggc tcg acc ggc
tcg att ggt tgc agc 48 Met Lys Gln Leu Thr Ile Leu Gly Ser Thr Gly
Ser Ile Gly Cys Ser 1 5 10 15 acg ctg gac gtg gtg cgc cat aat ccc
gaa cac ttc cgc gta gtt gcg 96 Thr Leu Asp Val Val Arg His Asn Pro
Glu His Phe Arg Val Val Ala 20 25 30 ctg gtg gca ggc aaa aat gtc
act cgc atg gta gaa cag tgc ctg gaa 144 Leu Val Ala Gly Lys Asn Val
Thr Arg Met Val Glu Gln Cys Leu Glu 35 40 45 ttc tct ccc cgc tat
gcc gta atg gac gat gaa gcg agt gcg aaa ctt 192 Phe Ser Pro Arg Tyr
Ala Val Met Asp Asp Glu Ala Ser Ala Lys Leu 50 55 60 ctt aaa acg
atg cta cag caa cag ggt agc cgc acc gaa gtc tta agt 240 Leu Lys Thr
Met Leu Gln Gln Gln Gly Ser Arg Thr Glu Val Leu Ser 65 70 75 80 ggg
caa caa gcc gct tgc gat atg gca gcg ctt gag gat gtt gat cag 288 Gly
Gln Gln Ala Ala Cys Asp Met Ala Ala Leu Glu Asp Val Asp Gln 85 90
95 gtg atg gca gcc att gtt ggc gct gct ggg ctg tta cct acg ctt gct
336 Val Met Ala Ala Ile Val Gly Ala Ala Gly Leu Leu Pro Thr Leu Ala
100 105 110 gcg atc cgc gcg ggt aaa acc att ttg ctg gcc aat aaa gaa
tca ctg 384 Ala Ile Arg Ala Gly Lys Thr Ile Leu Leu Ala Asn Lys Glu
Ser Leu 115 120 125 gtt acc tgc gga cgt ctg ttt atg gac gcc gta aag
cag agc aaa gcg 432 Val Thr Cys Gly Arg Leu Phe Met Asp Ala Val Lys
Gln Ser Lys Ala 130 135 140 caa ttg tta ccg gtc gat agc gaa cat aac
gcc att ttt cag agt tta 480 Gln Leu Leu Pro Val Asp Ser Glu His Asn
Ala Ile Phe Gln Ser Leu 145 150 155 160 ccg caa cct atc cag cat aat
ctg gga tac gct gac ctt gag caa aat 528 Pro Gln Pro Ile Gln His Asn
Leu Gly Tyr Ala Asp Leu Glu Gln Asn 165 170 175 ggc gtg gtg tcc att
tta ctt acc ggg tct ggt ggc cct ttc cgt gag 576 Gly Val Val Ser Ile
Leu Leu Thr Gly Ser Gly Gly Pro Phe Arg Glu 180 185 190 acg cca ttg
cgc gat ttg gca aca atg acg ccg gat caa gcc tgc cgt 624 Thr Pro Leu
Arg Asp Leu Ala Thr Met Thr Pro Asp Gln Ala Cys Arg 195 200 205 cat
ccg aac tgg tcg atg ggg cgt aaa att tct gtc gat tcg gct acc 672 His
Pro Asn Trp Ser Met Gly Arg Lys Ile Ser Val Asp Ser Ala Thr 210 215
220 atg atg aac aaa ggt ctg gaa tac att gaa gcg cgt tgg ctg ttt aac
720 Met Met Asn Lys Gly Leu Glu Tyr Ile Glu Ala Arg Trp Leu Phe Asn
225 230 235 240 gcc agc gcc agc cag atg gaa gtg ctg att cac ccg cag
tca gtg att 768 Ala Ser Ala Ser Gln Met Glu Val Leu Ile His Pro Gln
Ser Val Ile 245 250 255 cac tca atg gtg cgc tat cag gac ggc agt gtt
ctg gcg cag ctg ggg 816 His Ser Met Val Arg Tyr Gln Asp Gly Ser Val
Leu Ala Gln Leu Gly 260 265 270 gaa ccg gat atg gta cgc caa ttg ccc
aca cca tgg gca tgg ccg aat 864 Glu Pro Asp Met Val Arg Gln Leu Pro
Thr Pro Trp Ala Trp Pro Asn 275 280 285 cgc gtg aac tct ggc gtg aag
ccg ctc gat ttt tgc aaa cta agt gcg 912 Arg Val Asn Ser Gly Val Lys
Pro Leu Asp Phe Cys Lys Leu Ser Ala 290 295 300 ttg aca ttt gcc gca
ccg gat tat gat cgt tat cca tgc ctg aaa ctg 960 Leu Thr Phe Ala Ala
Pro Asp Tyr Asp Arg Tyr Pro Cys Leu Lys Leu 305 310 315 320 gcg atg
gag gcg ttc gaa caa ggc cag gca gcg acg aca gca ttg aat 1008 Ala
Met Glu Ala Phe Glu Gln Gly Gln Ala Ala Thr Thr Ala Leu Asn 325 330
335 gcc gca aac gaa atc acc gtt gct gct ttt ctt gcg caa caa atc cgc
1056 Ala Ala Asn Glu Ile Thr Val Ala Ala Phe Leu Ala Gln Gln Ile
Arg 340 345 350 ttt acg gat atc gct gcg ttg aat tta tcc gta ctg gaa
aaa atg gat 1104 Phe Thr Asp Ile Ala Ala Leu Asn Leu Ser Val Leu
Glu Lys Met Asp 355 360 365 atg cgc gaa cca caa tgt gtg gac gat gtg
tta tct gtt gat gcg aac 1152 Met Arg Glu Pro Gln Cys Val Asp Asp
Val Leu Ser Val Asp Ala Asn 370 375 380 gcg cgt gaa gtc gcc aga aaa
gag gtg atg cgt ctc gca agc 1194 Ala Arg Glu Val Ala Arg Lys Glu
Val Met Arg Leu Ala Ser 385 390 395 11 4390 DNA Escherichia coli
CDS (208)..(447) CDS (450)..(1346) CDS (1374)..(3233) CDS
(3344)..(4390) 11 atggcggcaa tggttcgttg gcaagcctta agcgacttgt
atagggaaaa atacagcagc 60 ccacacctgc ggctgcatcc aggcgcggaa
gtataccact aacatcgctt tgctgtgcac 120 atcaccttac cattgcgcgt
tatttgctat ttgccctgag tccgttacca tgacggggcg 180 aaaaatattg
agagtcagac attcatt atg ccg aag aaa aat gag gcg ccc gcc 234 Met Pro
Lys Lys Asn Glu Ala Pro Ala 1 5 agc ttt gaa aag gcg ctg agc gag ctg
gaa cag att gta acc cgt ctg 282 Ser Phe Glu Lys Ala Leu Ser Glu Leu
Glu Gln Ile Val Thr Arg Leu 10 15 20 25 gaa agt ggc gac ctg ccg ctg
gaa gag gcg ctg aac gag ttc gaa cgc 330 Glu Ser Gly Asp Leu Pro Leu
Glu Glu Ala Leu Asn Glu Phe Glu Arg 30 35 40 ggc gtg cag ctg gca
cgt cag ggg cag gcc aaa tta caa caa gcc gaa 378 Gly Val Gln Leu Ala
Arg Gln Gly Gln Ala Lys Leu Gln Gln Ala Glu 45 50 55 cag cgc gta
caa att ctg ctg tct gac aat gaa gac gcc tct cta acc 426 Gln Arg Val
Gln Ile Leu Leu Ser Asp Asn Glu Asp Ala Ser Leu Thr 60 65 70 cct
ttt aca ccg gac aat gag ta atg gac ttt ccg cag caa ctc gaa 473 Pro
Phe Thr Pro Asp Asn Glu Met Asp Phe Pro Gln Gln Leu Glu 75 80 1 5
gcc tgc gtt aag cag gcc aac cag gcg ctg agc cgt ttt atc gcc cca 521
Ala Cys Val Lys Gln Ala Asn Gln Ala Leu Ser Arg Phe Ile Ala Pro 10
15 20 ctg ccc ttt cag aac act ccc gtg gtc gaa acc atg cag tat ggc
gca 569 Leu Pro Phe Gln Asn Thr Pro Val Val Glu Thr Met Gln Tyr Gly
Ala 25 30 35 40 tta tta ggt ggt aag cgc ctg cga cct ttc ctg gtt tat
gcc acc ggt 617 Leu Leu Gly Gly Lys Arg Leu Arg Pro Phe Leu Val Tyr
Ala Thr Gly 45 50 55 cat atg ttc ggc gtt agc aca aac acg ctg gac
gca ccc gct gcc gcc 665 His Met Phe Gly Val Ser Thr Asn Thr Leu Asp
Ala Pro Ala Ala Ala 60 65
70 gtt gag tgt atc cac gct tac tca tta att cat gat gat tta ccg gca
713 Val Glu Cys Ile His Ala Tyr Ser Leu Ile His Asp Asp Leu Pro Ala
75 80 85 atg gat gat gac gat ctg cgt cgc ggt ttg cca acc tgc cat
gtg aag 761 Met Asp Asp Asp Asp Leu Arg Arg Gly Leu Pro Thr Cys His
Val Lys 90 95 100 ttt ggc gaa gca aac gcg att ctc gct ggc gac gct
tta caa acg ctg 809 Phe Gly Glu Ala Asn Ala Ile Leu Ala Gly Asp Ala
Leu Gln Thr Leu 105 110 115 120 gcg ttc tcg att tta agc gat gcc gat
atg ccg gaa gtg tcg gac cgc 857 Ala Phe Ser Ile Leu Ser Asp Ala Asp
Met Pro Glu Val Ser Asp Arg 125 130 135 gac aga att tcg atg att tct
gaa ctg gcg agc gcc agt ggt att gcc 905 Asp Arg Ile Ser Met Ile Ser
Glu Leu Ala Ser Ala Ser Gly Ile Ala 140 145 150 gga atg tgc ggt ggt
cag gca tta gat tta gac gcg gaa ggc aaa cac 953 Gly Met Cys Gly Gly
Gln Ala Leu Asp Leu Asp Ala Glu Gly Lys His 155 160 165 gta cct ctg
gac gcg ctt gag cgt att cat cgt cat aaa acc ggc gca 1001 Val Pro
Leu Asp Ala Leu Glu Arg Ile His Arg His Lys Thr Gly Ala 170 175 180
ttg att cgc gcc gcc gtt cgc ctt ggt gca tta agc gcc gga gat aaa
1049 Leu Ile Arg Ala Ala Val Arg Leu Gly Ala Leu Ser Ala Gly Asp
Lys 185 190 195 200 gga cgt cgt gct ctg ccg gta ctc gac aag tat gca
gag agc atc ggc 1097 Gly Arg Arg Ala Leu Pro Val Leu Asp Lys Tyr
Ala Glu Ser Ile Gly 205 210 215 ctt gcc ttc cag gtt cag gat gac atc
ctg gat gtg gtg gga gat act 1145 Leu Ala Phe Gln Val Gln Asp Asp
Ile Leu Asp Val Val Gly Asp Thr 220 225 230 gca acg ttg gga aaa cgc
cag ggt gcc gac cag caa ctt ggt aaa agt 1193 Ala Thr Leu Gly Lys
Arg Gln Gly Ala Asp Gln Gln Leu Gly Lys Ser 235 240 245 acc tac cct
gca ctt ctg ggt ctt gag caa gcc cgg aag aaa gcc cgg 1241 Thr Tyr
Pro Ala Leu Leu Gly Leu Glu Gln Ala Arg Lys Lys Ala Arg 250 255 260
gat ctg atc gac gat gcc cgt cag tcg ctg aaa caa ctg gct gaa cag
1289 Asp Leu Ile Asp Asp Ala Arg Gln Ser Leu Lys Gln Leu Ala Glu
Gln 265 270 275 280 tca ctc gat acc tcg gca ctg gaa gcg cta gcg gac
tac atc atc cag 1337 Ser Leu Asp Thr Ser Ala Leu Glu Ala Leu Ala
Asp Tyr Ile Ile Gln 285 290 295 cgt aat aaa taaacaataa gtattaatag
gcccctg atg agt ttt gat att gcc 1391 Arg Asn Lys Met Ser Phe Asp
Ile Ala 1 5 aaa tac ccg acc ctg gca ctg gtc gac tcc acc cag gag tta
cga ctg 1439 Lys Tyr Pro Thr Leu Ala Leu Val Asp Ser Thr Gln Glu
Leu Arg Leu 10 15 20 ttg ccg aaa gag agt tta ccg aaa ctc tgc gac
gaa ctg cgc cgc tat 1487 Leu Pro Lys Glu Ser Leu Pro Lys Leu Cys
Asp Glu Leu Arg Arg Tyr 25 30 35 tta ctc gac agc gtg agc cgt tcc
agc ggg cac ttc gcc tcc ggg ctg 1535 Leu Leu Asp Ser Val Ser Arg
Ser Ser Gly His Phe Ala Ser Gly Leu 40 45 50 ggc acg gtc gaa ctg
acc gtg gcg ctg cac tat gtc tac aac acc ccg 1583 Gly Thr Val Glu
Leu Thr Val Ala Leu His Tyr Val Tyr Asn Thr Pro 55 60 65 70 ttt gac
caa ttg att tgg gat gtg ggg cat cag gct tat ccg cat aaa 1631 Phe
Asp Gln Leu Ile Trp Asp Val Gly His Gln Ala Tyr Pro His Lys 75 80
85 att ttg acc gga cgc cgc gac aaa atc ggc acc atc cgt cag aaa ggc
1679 Ile Leu Thr Gly Arg Arg Asp Lys Ile Gly Thr Ile Arg Gln Lys
Gly 90 95 100 ggt ctg cac ccg ttc ccg tgg cgc ggc gaa agc gaa tat
gac gta tta 1727 Gly Leu His Pro Phe Pro Trp Arg Gly Glu Ser Glu
Tyr Asp Val Leu 105 110 115 agc gtc ggg cat tca tca acc tcc atc agt
gcc gga att ggt att gcg 1775 Ser Val Gly His Ser Ser Thr Ser Ile
Ser Ala Gly Ile Gly Ile Ala 120 125 130 gtt gct gcc gaa aaa gaa ggc
aaa aat cgc cgc acc gtc tgt gtc att 1823 Val Ala Ala Glu Lys Glu
Gly Lys Asn Arg Arg Thr Val Cys Val Ile 135 140 145 150 ggc gat ggc
gcg att acc gca ggc atg gcg ttt gaa gcg atg aat cac 1871 Gly Asp
Gly Ala Ile Thr Ala Gly Met Ala Phe Glu Ala Met Asn His 155 160 165
gcg ggc gat atc cgt cct gat atg ctg gtg att ctc aac gac aat gaa
1919 Ala Gly Asp Ile Arg Pro Asp Met Leu Val Ile Leu Asn Asp Asn
Glu 170 175 180 atg tcg att tcc gaa aat gtc ggc gcg ctc aac aac cat
ctg gca cag 1967 Met Ser Ile Ser Glu Asn Val Gly Ala Leu Asn Asn
His Leu Ala Gln 185 190 195 ctg ctt tcc ggt aag ctt tac tct tca ctg
cgc gaa ggc ggg aaa aaa 2015 Leu Leu Ser Gly Lys Leu Tyr Ser Ser
Leu Arg Glu Gly Gly Lys Lys 200 205 210 gtt ttc tct ggc gtg ccg cca
att aaa gag ctg ctc aaa cgc acc gaa 2063 Val Phe Ser Gly Val Pro
Pro Ile Lys Glu Leu Leu Lys Arg Thr Glu 215 220 225 230 gaa cat att
aaa ggc atg gta gtg cct ggc acg ttg ttt gaa gag ctg 2111 Glu His
Ile Lys Gly Met Val Val Pro Gly Thr Leu Phe Glu Glu Leu 235 240 245
ggc ttt aac tac atc ggc ccg gtg gac ggt cac gat gtg ctg ggg ctt
2159 Gly Phe Asn Tyr Ile Gly Pro Val Asp Gly His Asp Val Leu Gly
Leu 250 255 260 atc acc acg cta aag aac atg cgc gac ctg aaa ggc ccg
cag ttc ctg 2207 Ile Thr Thr Leu Lys Asn Met Arg Asp Leu Lys Gly
Pro Gln Phe Leu 265 270 275 cat atc atg acc aaa aaa ggt cgt ggt tat
gaa ccg gca gaa aaa gac 2255 His Ile Met Thr Lys Lys Gly Arg Gly
Tyr Glu Pro Ala Glu Lys Asp 280 285 290 ccg atc act ttc cac gcc gtg
cct aaa ttt gat ccc tcc agc ggt tgt 2303 Pro Ile Thr Phe His Ala
Val Pro Lys Phe Asp Pro Ser Ser Gly Cys 295 300 305 310 ttg ccg aaa
agt agc ggc ggt ttg ccg agc tat tca aaa atc ttt ggc 2351 Leu Pro
Lys Ser Ser Gly Gly Leu Pro Ser Tyr Ser Lys Ile Phe Gly 315 320 325
gac tgg ttg tgc gaa acg gca gcg aaa gac aac aag ctg atg gcg att
2399 Asp Trp Leu Cys Glu Thr Ala Ala Lys Asp Asn Lys Leu Met Ala
Ile 330 335 340 act ccg gcg atg cgt gaa ggt tcc ggc atg gtc gag ttt
tca cgt aaa 2447 Thr Pro Ala Met Arg Glu Gly Ser Gly Met Val Glu
Phe Ser Arg Lys 345 350 355 ttc ccg gat cgc tac ttc gac gtg gca att
gcc gag caa cac gcg gtg 2495 Phe Pro Asp Arg Tyr Phe Asp Val Ala
Ile Ala Glu Gln His Ala Val 360 365 370 acc ttt gct gcg ggt ctg gcg
att ggt ggg tac aaa ccc att gtc gcg 2543 Thr Phe Ala Ala Gly Leu
Ala Ile Gly Gly Tyr Lys Pro Ile Val Ala 375 380 385 390 att tac tcc
act ttc ctg caa cgc gcc tat gat cag gtg ctg cat gac 2591 Ile Tyr
Ser Thr Phe Leu Gln Arg Ala Tyr Asp Gln Val Leu His Asp 395 400 405
gtg gcg att caa aag ctt ccg gtc ctg ttc gcc atc gac cgc gcg ggc
2639 Val Ala Ile Gln Lys Leu Pro Val Leu Phe Ala Ile Asp Arg Ala
Gly 410 415 420 att gtt ggt gct gac ggt caa acc cat cag ggt gct ttt
gat ctc tct 2687 Ile Val Gly Ala Asp Gly Gln Thr His Gln Gly Ala
Phe Asp Leu Ser 425 430 435 tac ctg cgc tgc ata ccg gaa atg gtc att
atg acc ccg agc gat gaa 2735 Tyr Leu Arg Cys Ile Pro Glu Met Val
Ile Met Thr Pro Ser Asp Glu 440 445 450 aac gaa tgt cgc cag atg ctc
tat acc ggc tat cac tat aac gat ggc 2783 Asn Glu Cys Arg Gln Met
Leu Tyr Thr Gly Tyr His Tyr Asn Asp Gly 455 460 465 470 ccg tca gcg
gtg cgc tac ccg cgt ggc aac gcg gtc ggc gtg gaa ctg 2831 Pro Ser
Ala Val Arg Tyr Pro Arg Gly Asn Ala Val Gly Val Glu Leu 475 480 485
acg ccg ctg gaa aaa cta cca att ggc aaa ggc att gtg aag cgt cgt
2879 Thr Pro Leu Glu Lys Leu Pro Ile Gly Lys Gly Ile Val Lys Arg
Arg 490 495 500 ggc gag aaa ctg gcg atc ctt aac ttt ggt acg ctg atg
cca gaa gcg 2927 Gly Glu Lys Leu Ala Ile Leu Asn Phe Gly Thr Leu
Met Pro Glu Ala 505 510 515 gcg aaa gtc gcc gaa tcg ctg aac gcc acg
ctg gtc gat atg cgt ttt 2975 Ala Lys Val Ala Glu Ser Leu Asn Ala
Thr Leu Val Asp Met Arg Phe 520 525 530 gtg aaa ccg ctt gat gaa gcg
tta att ctg gaa atg gcc gcc agc cat 3023 Val Lys Pro Leu Asp Glu
Ala Leu Ile Leu Glu Met Ala Ala Ser His 535 540 545 550 gaa gcg ctg
gtc acc gta gaa gaa aac gcc att atg ggc ggc gca ggc 3071 Glu Ala
Leu Val Thr Val Glu Glu Asn Ala Ile Met Gly Gly Ala Gly 555 560 565
agc ggc gtg aac gaa gtg ctg atg gcc cat cgt aaa cca gta ccc gtg
3119 Ser Gly Val Asn Glu Val Leu Met Ala His Arg Lys Pro Val Pro
Val 570 575 580 ctg aac att ggc ctg ccg gac ttc ttt att ccg caa gga
act cag gaa 3167 Leu Asn Ile Gly Leu Pro Asp Phe Phe Ile Pro Gln
Gly Thr Gln Glu 585 590 595 gaa atg cgc gcc gaa ctc ggc ctc gat gcc
gct ggt atg gaa gcc aaa 3215 Glu Met Arg Ala Glu Leu Gly Leu Asp
Ala Ala Gly Met Glu Ala Lys 600 605 610 atc aag gcc tgg ctg gca
taatccctac tccactcctg ctatgcttaa 3263 Ile Lys Ala Trp Leu Ala 615
620 gaaattattc atagactcta aataattcga gttgcaggaa ggcggcaaac
gagtgaagcc 3323 ccaggagctt acataagtaa gtg act ggg gtg aac gaa tgc
agc cgc agc aca 3376 Val Thr Gly Val Asn Glu Cys Ser Arg Ser Thr 1
5 10 tgc aac ttg aag tat gac gag tat agc agg agt ggc agc atg caa
tac 3424 Cys Asn Leu Lys Tyr Asp Glu Tyr Ser Arg Ser Gly Ser Met
Gln Tyr 15 20 25 aac ccc tta gga aaa acc gac ctt cgc gtt tcc cga
ctt tgc ctc ggc 3472 Asn Pro Leu Gly Lys Thr Asp Leu Arg Val Ser
Arg Leu Cys Leu Gly 30 35 40 tgt atg acc ttt ggc gag cca gat cgc
ggt aat cac gca tgg aca ctg 3520 Cys Met Thr Phe Gly Glu Pro Asp
Arg Gly Asn His Ala Trp Thr Leu 45 50 55 ccg gaa gaa agc agc cgt
ccc ata att aaa cgt gca ctg gaa ggc ggc 3568 Pro Glu Glu Ser Ser
Arg Pro Ile Ile Lys Arg Ala Leu Glu Gly Gly 60 65 70 75 ata aat ttc
ttt gat acc gcc aac agt tat tct gac ggc agc agc gaa 3616 Ile Asn
Phe Phe Asp Thr Ala Asn Ser Tyr Ser Asp Gly Ser Ser Glu 80 85 90
gag atc gtc ggt cgc gca ctg cgg gat ttc gcc cgt cgt gaa gac gtg
3664 Glu Ile Val Gly Arg Ala Leu Arg Asp Phe Ala Arg Arg Glu Asp
Val 95 100 105 gtc gtt gcg acc aaa gtg ttc cat cgc gtt ggt gat tta
ccg gaa gga 3712 Val Val Ala Thr Lys Val Phe His Arg Val Gly Asp
Leu Pro Glu Gly 110 115 120 tta tcc cgt gcg caa att ttg cgc tct atc
gac gac agc ctg cga cgt 3760 Leu Ser Arg Ala Gln Ile Leu Arg Ser
Ile Asp Asp Ser Leu Arg Arg 125 130 135 ctc ggc atg gat tat gtc gat
atc ctg caa att cat cgc tgg gat tac 3808 Leu Gly Met Asp Tyr Val
Asp Ile Leu Gln Ile His Arg Trp Asp Tyr 140 145 150 155 aac acg ccg
atc gaa gag acg ctg gaa gcc ctc aac gac gtg gta aaa 3856 Asn Thr
Pro Ile Glu Glu Thr Leu Glu Ala Leu Asn Asp Val Val Lys 160 165 170
gcc ggg aaa gcg cgt tat atc ggc gcg tca tca atg cac gct tcg cag
3904 Ala Gly Lys Ala Arg Tyr Ile Gly Ala Ser Ser Met His Ala Ser
Gln 175 180 185 ttt gct cag gca ctg gaa ctc caa aaa cag cac ggc tgg
gcg cag ttt 3952 Phe Ala Gln Ala Leu Glu Leu Gln Lys Gln His Gly
Trp Ala Gln Phe 190 195 200 gtc agt atg cag gat cac tac aat ctg att
tat cgt gaa gaa gag cgc 4000 Val Ser Met Gln Asp His Tyr Asn Leu
Ile Tyr Arg Glu Glu Glu Arg 205 210 215 gag atg cta cca ctg tgt tat
cag gag ggc gtg gcg gta att cca tgg 4048 Glu Met Leu Pro Leu Cys
Tyr Gln Glu Gly Val Ala Val Ile Pro Trp 220 225 230 235 agc ccg ctg
gca agg ggc cgt ctg acg cgt ccg tgg gga gaa act acc 4096 Ser Pro
Leu Ala Arg Gly Arg Leu Thr Arg Pro Trp Gly Glu Thr Thr 240 245 250
gca cga ctg gtg tct gat gag gtg ggg aaa aat ctc tat aaa gaa agc
4144 Ala Arg Leu Val Ser Asp Glu Val Gly Lys Asn Leu Tyr Lys Glu
Ser 255 260 265 gat gaa aat gac gcg cag atc gca gag cgg tta aca ggc
gtc agt gaa 4192 Asp Glu Asn Asp Ala Gln Ile Ala Glu Arg Leu Thr
Gly Val Ser Glu 270 275 280 gaa ctg ggg gcg aca cga gca caa gtt gcg
ctg gcc tgg ttg ttg agt 4240 Glu Leu Gly Ala Thr Arg Ala Gln Val
Ala Leu Ala Trp Leu Leu Ser 285 290 295 aaa ccg ggc att gcc gca ccg
att atc gga act tcg cgc gaa gaa cag 4288 Lys Pro Gly Ile Ala Ala
Pro Ile Ile Gly Thr Ser Arg Glu Glu Gln 300 305 310 315 ctt gat gag
cta ttg aac gcg gtg gat atc act ttg aag ccg gaa cag 4336 Leu Asp
Glu Leu Leu Asn Ala Val Asp Ile Thr Leu Lys Pro Glu Gln 320 325 330
att gcc gaa ctg gaa acg ccg tat aaa ccg cat cct gtc gta gga ttt
4384 Ile Ala Glu Leu Glu Thr Pro Tyr Lys Pro His Pro Val Val Gly
Phe 335 340 345 aaa taa 4390 Lys 12 33 DNA Artificial Sequence
Description of Artificial SequenceSynthetic DNA 12 ccggatccat
ggcggcaatg gttcgttggc aag 33 13 34 DNA Artificial Sequence
Description of Artificial SequenceSynthetic DNA 13 ccgaattctt
atttaaatcc tacgacagga tgcg 34 14 33 DNA Artificial Sequence
Description of Artificial SequenceSynthetic DNA 14 ccggatccat
gagttttgat attgccaaat acc 33 15 33 DNA Artificial Sequence
Description of Artificial SequenceSynthetic DNA 15 ccgaattctt
atgccagcca ggccttgatt ttg 33 16 33 DNA Artificial Sequence
Description of Artificial SequenceSynthetic DNA 16 ccgaattctt
actcattgtc cggtgtaaaa ggg 33 17 33 DNA Artificial Sequence
Description of Artificial SequenceSynthetic DNA 17 ccggatccat
ggactttccg cagcaactcg aag 33 18 33 DNA Artificial Sequence
Description of Artificial SequenceSynthetic DNA 18 ccgaattctt
atttattacg ctggatgatg tag 33 19 33 DNA Artificial Sequence
Description of Artificial SequenceSynthetic DNA 19 ccggatccta
atccctactc cactcctgct atg 33 20 30 DNA Artificial Sequence
Description of Artificial SequenceSynthetic DNA 20 gggggatcca
agcaactcac cattctgggc 30 21 30 DNA Artificial Sequence Description
of Artificial SequenceSynthetic DNA 21 gggggatccg cttgcgagac
gcatcacctc 30 22 32 DNA Artificial Sequence Description of
Artificial SequenceSynthetic DNA 22 gggggatcca gttttgatat
tgccaaatac cc 32 23 32 DNA Artificial Sequence Description of
Artificial SequenceSynthetic DNA 23 gggggatcct gccagccagg
ccttgatttt gg 32 24 30 DNA Artificial Sequence Description of
Artificial SequenceSynthetic DNA 24 gggggatccg agcaactcac
cattctgggc 30 25 30 DNA Artificial Sequence Description of
Artificial SequenceSynthetic DNA 25 gggggatccg cttgcgagac
gcatcacctc 30 26 637 PRT Rhodobacter sphaeroides 26 Met Thr Asp Arg
Pro Cys Thr Pro Thr Leu Asp Arg Val Thr Leu Pro 1 5 10 15 Val Asp
Met Lys Gly Leu Thr Asp Arg Glu Leu Arg Ser Leu Ala Asp 20 25 30
Glu Leu Arg Ala Glu Thr Ile Ser Ala Val Ser Val Thr Gly Gly His 35
40 45 Leu Gly Ala Gly Leu Gly Val Val Glu Leu Thr Val Ala Leu His
Ala 50 55 60 Val Phe Asp Ala Pro Arg Asp Lys Ile Ile Trp Asp Val
Gly His Gln 65 70 75 80 Cys Tyr Pro His Lys Ile Leu Thr Gly Arg Arg
Asp Arg Ile Arg Thr 85 90 95 Leu Arg Gln Gly Gly Gly Leu Ser Gly
Phe Thr Lys Arg Ser Glu Ser 100 105 110 Pro Tyr Asp Cys Phe Gly Ala
Gly His Ser Ser Thr Ser Ile Ser Ala 115 120 125 Ala Val Gly Phe Ala
Ala Ala Arg Glu Met Gly Gly Asp Thr Gly Asp 130 135 140 Ala Val Ala
Val Ile Gly Asp Gly Ser Met Ser Ala Gly Met Ala Phe 145 150 155 160
Glu Ala Leu Asn His Gly Gly His Leu Lys Asn Arg Val Ile
Val Ile 165 170 175 Leu Asn Asp Asn Glu Met Ser Ile Ala Pro Pro Val
Gly Ala Leu Ser 180 185 190 Ser Tyr Leu Ser Arg Leu Tyr Ala Gly Ala
Pro Phe Gln Asp Phe Lys 195 200 205 Ala Ala Ala Lys Gly Ala Leu Gly
Leu Leu Pro Glu Pro Phe Gln Glu 210 215 220 Gly Ala Arg Arg Ala Lys
Glu Met Leu Lys Ser Val Thr Val Gly Gly 225 230 235 240 Thr Leu Phe
Glu Glu Leu Gly Phe Ser Tyr Val Gly Pro Ile Asp Gly 245 250 255 His
Asp Leu Asp Gln Leu Leu Pro Val Leu Arg Thr Val Lys Gln Arg 260 265
270 Ala His Ala Pro Val Leu Ile His Val Ile Thr Lys Lys Gly Arg Gly
275 280 285 Tyr Ala Pro Ala Glu Ala Ala Arg Asp Arg Gly His Ala Thr
Asn Lys 290 295 300 Phe Asn Val Leu Thr Gly Ala Gln Val Lys Pro Val
Ser Asn Ala Pro 305 310 315 320 Ser Tyr Thr Lys Val Phe Ala Gln Ser
Leu Ile Lys Glu Ala Glu Val 325 330 335 Asp Glu Arg Ile Cys Ala Val
Thr Ala Ala Met Pro Asp Gly Thr Gly 340 345 350 Leu Asn Leu Phe Gly
Glu Arg Phe Pro Lys Arg Thr Phe Asp Val Gly 355 360 365 Ile Ala Glu
Gln His Ala Val Thr Phe Ser Ala Ala Leu Ala Ala Gly 370 375 380 Gly
Met Arg Pro Phe Cys Ala Ile Tyr Ser Thr Phe Leu Gln Arg Gly 385 390
395 400 Tyr Asp Gln Ile Val His Asp Val Ala Ile Gln Arg Leu Pro Val
Arg 405 410 415 Phe Ala Ile Asp Arg Ala Gly Leu Val Gly Ala Asp Gly
Ala Thr His 420 425 430 Ala Gly Ser Phe Asp Val Ala Phe Leu Ser Asn
Leu Pro Gly Ile Val 435 440 445 Val Met Ala Ala Ala Asp Glu Ala Glu
Leu Val His Met Val Ala Thr 450 455 460 Ala Ala Ala His Asp Glu Gly
Pro Ile Ala Phe Arg Tyr Pro Arg Gly 465 470 475 480 Asp Gly Val Gly
Val Glu Met Pro Val Lys Gly Val Pro Leu Gln Ile 485 490 495 Gly Arg
Gly Arg Val Val Arg Glu Gly Thr Arg Ile Ala Leu Leu Ser 500 505 510
Phe Gly Thr Arg Leu Ala Glu Val Gln Val Ala Ala Glu Ala Leu Arg 515
520 525 Ala Arg Gly Ile Ser Pro Thr Val Ala Asp Ala Arg Phe Ala Lys
Pro 530 535 540 Leu Asp Arg Asp Leu Ile Leu Gln Leu Ala Ala His His
Glu Ala Leu 545 550 555 560 Ile Thr Ile Glu Glu Gly Ala Ile Gly Gly
Phe Gly Ser His Val Ala 565 570 575 Gln Leu Leu Ala Glu Ala Gly Val
Phe Asp Arg Gly Phe Arg Tyr Arg 580 585 590 Ser Met Val Leu Pro Asp
Thr Phe Ile Asp His Asn Ser Ala Glu Val 595 600 605 Met Tyr Ala Thr
Ala Gly Leu Asn Ala Ala Asp Ile Glu Arg Lys Ala 610 615 620 Leu Glu
Thr Leu Gly Val Glu Val Leu Ala Arg Arg Ala 625 630 635 27 1911 DNA
Rhodobacter sphaeroides CDS (1)..(1911) 27 atg acc gac aga ccc tgc
acg ccg acg ctc gac cgg gtg acg ctc ccg 48 Met Thr Asp Arg Pro Cys
Thr Pro Thr Leu Asp Arg Val Thr Leu Pro 1 5 10 15 gtg gac atg aag
ggc ctc acg gac cgt gag ctg cgc tcg ctg gcc gac 96 Val Asp Met Lys
Gly Leu Thr Asp Arg Glu Leu Arg Ser Leu Ala Asp 20 25 30 gag ctg
cgg gcc gaa acg atc tcg gcc gtg tcg gtg acg ggc ggg cat 144 Glu Leu
Arg Ala Glu Thr Ile Ser Ala Val Ser Val Thr Gly Gly His 35 40 45
ctg ggc gca ggc ctc ggc gtg gtg gag ttg acg gtt gcg ctg cat gcg 192
Leu Gly Ala Gly Leu Gly Val Val Glu Leu Thr Val Ala Leu His Ala 50
55 60 gtc ttc gat gcg ccg cgc gac aag atc atc tgg gac gtg ggc cac
cag 240 Val Phe Asp Ala Pro Arg Asp Lys Ile Ile Trp Asp Val Gly His
Gln 65 70 75 80 tgc tac ccc cac aag atc ctg acc ggg cgg cgc gac cgc
atc cgc aca 288 Cys Tyr Pro His Lys Ile Leu Thr Gly Arg Arg Asp Arg
Ile Arg Thr 85 90 95 ctg cgg cag ggc ggg ggt ctc tcg ggc ttc acc
aag cgc tcc gag agc 336 Leu Arg Gln Gly Gly Gly Leu Ser Gly Phe Thr
Lys Arg Ser Glu Ser 100 105 110 ccc tac gac tgt ttc ggc gcg ggc cat
tcc tcg acc tcg atc tcg gcc 384 Pro Tyr Asp Cys Phe Gly Ala Gly His
Ser Ser Thr Ser Ile Ser Ala 115 120 125 gcg gtg ggc ttt gcc gcg gcg
cgc gag atg ggc ggc gac acg ggc gac 432 Ala Val Gly Phe Ala Ala Ala
Arg Glu Met Gly Gly Asp Thr Gly Asp 130 135 140 gcg gtg gcg gtg atc
ggc gat ggc tcg atg tcg gcc ggc atg gcc ttc 480 Ala Val Ala Val Ile
Gly Asp Gly Ser Met Ser Ala Gly Met Ala Phe 145 150 155 160 gag gcg
ctg aac cac ggc ggg cac ctg aag aac cgg gtg atc gtg atc 528 Glu Ala
Leu Asn His Gly Gly His Leu Lys Asn Arg Val Ile Val Ile 165 170 175
ctg aac gac aat gag atg agc atc gcg ccg ccg gtg ggg gcg ctg tcg 576
Leu Asn Asp Asn Glu Met Ser Ile Ala Pro Pro Val Gly Ala Leu Ser 180
185 190 tcc tat ctc tcg cgg ctc tat gcg ggc gcg ccg ttc cag gac ttc
aag 624 Ser Tyr Leu Ser Arg Leu Tyr Ala Gly Ala Pro Phe Gln Asp Phe
Lys 195 200 205 gcg gcc gcc aag gga gcg ctc ggg ctt ctg ccc gaa ccg
ttc cag gag 672 Ala Ala Ala Lys Gly Ala Leu Gly Leu Leu Pro Glu Pro
Phe Gln Glu 210 215 220 ggc gcg cgc cgc gcc aag gag atg ctg aag agc
gtc acc gtc ggc ggc 720 Gly Ala Arg Arg Ala Lys Glu Met Leu Lys Ser
Val Thr Val Gly Gly 225 230 235 240 acg ctc ttc gag gag ctg ggt ttc
tcc tat gtc ggc ccg atc gac ggg 768 Thr Leu Phe Glu Glu Leu Gly Phe
Ser Tyr Val Gly Pro Ile Asp Gly 245 250 255 cac gat ctc gac cag ctt
ctg ccg gtg ctg cgg acc gtc aag cag cgg 816 His Asp Leu Asp Gln Leu
Leu Pro Val Leu Arg Thr Val Lys Gln Arg 260 265 270 gcg cat gcg ccg
gtg ctg atc cat gtc atc acc aag aag ggc agg ggc 864 Ala His Ala Pro
Val Leu Ile His Val Ile Thr Lys Lys Gly Arg Gly 275 280 285 tat gct
ccg gcc gag gcc gcg cgc gac cgc ggc cat gcc acg aac aag 912 Tyr Ala
Pro Ala Glu Ala Ala Arg Asp Arg Gly His Ala Thr Asn Lys 290 295 300
ttc aac gtc ctg acc ggc gcg cag gtg aag ccg gtc tcg aac gcc ccc 960
Phe Asn Val Leu Thr Gly Ala Gln Val Lys Pro Val Ser Asn Ala Pro 305
310 315 320 tcc tac acc aag gtc ttc gcc cag agc ctc atc aag gag gcc
gag gtc 1008 Ser Tyr Thr Lys Val Phe Ala Gln Ser Leu Ile Lys Glu
Ala Glu Val 325 330 335 gac gag cgg atc tgc gcg gtg acg gcc gcc atg
ccg gac ggg acg ggg 1056 Asp Glu Arg Ile Cys Ala Val Thr Ala Ala
Met Pro Asp Gly Thr Gly 340 345 350 ctc aac ctc ttc ggc gag cgg ttt
ccg aag cgc acc ttc gac gtg ggc 1104 Leu Asn Leu Phe Gly Glu Arg
Phe Pro Lys Arg Thr Phe Asp Val Gly 355 360 365 atc gcg gaa cag cat
gcg gtg acc ttc tcg gcg gcg ctt gcg gca ggc 1152 Ile Ala Glu Gln
His Ala Val Thr Phe Ser Ala Ala Leu Ala Ala Gly 370 375 380 ggc atg
cgg ccc ttc tgc gcg atc tat tcc acc ttc ctc cag cgc ggc 1200 Gly
Met Arg Pro Phe Cys Ala Ile Tyr Ser Thr Phe Leu Gln Arg Gly 385 390
395 400 tac gac cag atc gtg cat gac gtg gcg atc cag cgc ctg ccg gtg
cgc 1248 Tyr Asp Gln Ile Val His Asp Val Ala Ile Gln Arg Leu Pro
Val Arg 405 410 415 ttc gcc atc gat cgc gcg ggc ctc gtg ggg gcg gac
ggc gcc acc cat 1296 Phe Ala Ile Asp Arg Ala Gly Leu Val Gly Ala
Asp Gly Ala Thr His 420 425 430 gcg ggc tcg ttc gac gtg gcc ttc ctg
tcg aac ctg ccc ggc atc gtg 1344 Ala Gly Ser Phe Asp Val Ala Phe
Leu Ser Asn Leu Pro Gly Ile Val 435 440 445 gtg atg gcc gcc gcc gac
gag gcc gag ctc gtc cat atg gtg gcc acc 1392 Val Met Ala Ala Ala
Asp Glu Ala Glu Leu Val His Met Val Ala Thr 450 455 460 gcc gcc gcc
cat gac gaa ggg ccc atc gcc ttc cgc tac ccg cgc ggc 1440 Ala Ala
Ala His Asp Glu Gly Pro Ile Ala Phe Arg Tyr Pro Arg Gly 465 470 475
480 gac ggc gtg ggg gtc gag atg ccg gtg aag ggc gtg ccg ctc cag atc
1488 Asp Gly Val Gly Val Glu Met Pro Val Lys Gly Val Pro Leu Gln
Ile 485 490 495 ggc cgc ggc cgt gtg gtg cgc gag ggc acg cga atc gcg
ctt ttg tcc 1536 Gly Arg Gly Arg Val Val Arg Glu Gly Thr Arg Ile
Ala Leu Leu Ser 500 505 510 ttc ggc acc cgt ctg gcc gag gtg cag gtg
gcc gcc gag gcg ctg cgt 1584 Phe Gly Thr Arg Leu Ala Glu Val Gln
Val Ala Ala Glu Ala Leu Arg 515 520 525 gcg cgc ggg atc tct ccc acg
gtt gcg gat gcg cgc ttt gca aag ccg 1632 Ala Arg Gly Ile Ser Pro
Thr Val Ala Asp Ala Arg Phe Ala Lys Pro 530 535 540 ctc gac cgg gat
ctg atc ctg cag ctc gcg gcc cat cac gag gcg ctt 1680 Leu Asp Arg
Asp Leu Ile Leu Gln Leu Ala Ala His His Glu Ala Leu 545 550 555 560
atc acc atc gag gag ggc gcc atc ggc ggt ttc ggc agc cat gtg gcg
1728 Ile Thr Ile Glu Glu Gly Ala Ile Gly Gly Phe Gly Ser His Val
Ala 565 570 575 cag ctt ctg gcc gag gcc ggg gtc ttc gac cgc ggc ttc
cgg tat cgc 1776 Gln Leu Leu Ala Glu Ala Gly Val Phe Asp Arg Gly
Phe Arg Tyr Arg 580 585 590 tcg atg gtg ctg ccc gac acg ttc atc gac
cac aac agc gcg gag gtg 1824 Ser Met Val Leu Pro Asp Thr Phe Ile
Asp His Asn Ser Ala Glu Val 595 600 605 atg tat gcc acc gcc ggg ctg
aat gcg gcc gac ata gag cgg aag gcg 1872 Met Tyr Ala Thr Ala Gly
Leu Asn Ala Ala Asp Ile Glu Arg Lys Ala 610 615 620 ctg gag acg ctg
ggg gtg gag gtc ctc gcc cgc cgc gcc 1911 Leu Glu Thr Leu Gly Val
Glu Val Leu Ala Arg Arg Ala 625 630 635 28 648 PRT Rhodobacter
sphaeroides 28 Met Thr Asn Pro Thr Pro Arg Pro Glu Thr Pro Leu Leu
Asp Arg Val 1 5 10 15 Cys Cys Pro Ala Asp Met Lys Ala Leu Ser Asp
Ala Glu Leu Glu Arg 20 25 30 Leu Ala Asp Glu Val Arg Ser Glu Val
Ile Ser Val Val Ala Glu Thr 35 40 45 Gly Gly His Leu Gly Ser Ser
Leu Gly Val Val Glu Leu Thr Val Ala 50 55 60 Leu His Ala Val Phe
Asn Thr Pro Thr Asp Lys Leu Val Trp Asp Val 65 70 75 80 Gly His Gln
Cys Tyr Pro His Lys Ile Leu Thr Gly Arg Arg Glu Gln 85 90 95 Met
Arg Thr Leu Arg Gln Lys Gly Gly Leu Ser Gly Phe Thr Lys Arg 100 105
110 Ser Glu Ser Ala Tyr Asp Pro Phe Gly Ala Ala His Ser Ser Thr Ser
115 120 125 Ile Ser Ala Ala Leu Gly Phe Ala Met Gly Arg Glu Leu Gly
Gln Pro 130 135 140 Val Gly Asp Thr Ile Ala Val Ile Gly Asp Gly Ser
Ile Thr Ala Gly 145 150 155 160 Met Ala Tyr Glu Ala Leu Asn His Ala
Gly His Leu Asn Lys Arg Leu 165 170 175 Phe Val Ile Leu Asn Asp Asn
Asp Met Ser Ile Ala Pro Pro Val Gly 180 185 190 Ala Leu Ala Arg Tyr
Leu Val Asn Leu Ser Ser Lys Ala Pro Phe Ala 195 200 205 Thr Leu Arg
Ala Ala Ala Asp Gly Leu Glu Ala Ser Leu Pro Gly Pro 210 215 220 Leu
Arg Asp Gly Ala Arg Arg Ala Arg Gln Leu Val Thr Gly Met Pro 225 230
235 240 Gly Gly Gly Thr Leu Phe Glu Glu Leu Gly Phe Thr Tyr Val Gly
Pro 245 250 255 Ile Asp Gly His Asp Met Glu Ala Leu Leu Gln Thr Leu
Arg Ala Ala 260 265 270 Arg Ala Arg Thr Thr Gly Pro Val Leu Ile His
Val Val Thr Lys Lys 275 280 285 Gly Lys Gly Tyr Ala Pro Ala Glu Asn
Ala Pro Asp Lys Tyr His Gly 290 295 300 Val Asn Lys Phe Asp Pro Val
Thr Gly Glu Gln Lys Lys Ser Val Ala 305 310 315 320 Asn Ala Pro Asn
Tyr Thr Lys Val Phe Gly Ser Thr Leu Thr Glu Glu 325 330 335 Ala Ala
Arg Asp Pro Arg Ile Val Ala Ile Thr Ala Ala Met Pro Ser 340 345 350
Gly Thr Gly Val Asp Ile Met Gln Lys Arg Phe Pro Asn Arg Val Phe 355
360 365 Asp Val Gly Ile Ala Glu Gln His Ala Val Thr Phe Ala Ala Gly
Leu 370 375 380 Ala Gly Ala Gly Met Lys Pro Phe Cys Ala Ile Tyr Ser
Ser Phe Leu 385 390 395 400 Gln Arg Gly Tyr Asp Gln Ile Ala His Asp
Val Ala Leu Gln Asn Leu 405 410 415 Pro Val Arg Phe Val Ile Asp Arg
Ala Gly Leu Val Gly Ala Asp Gly 420 425 430 Ala Thr His Ala Gly Ala
Phe Asp Val Gly Phe Leu Thr Ser Leu Pro 435 440 445 Asn Met Thr Val
Met Ala Ala Ala Asp Glu Ala Glu Leu Ile His Met 450 455 460 Ile Ala
Thr Ala Val Ala Phe Asp Glu Gly Pro Ile Ala Phe Arg Phe 465 470 475
480 Pro Arg Gly Glu Gly Val Gly Val Glu Met Pro Glu Arg Gly Thr Val
485 490 495 Leu Glu Pro Gly Arg Gly Arg Val Val Arg Glu Gly Thr Asp
Val Ala 500 505 510 Ile Leu Ser Phe Gly Ala His Leu His Glu Ala Leu
Gln Ala Ala Lys 515 520 525 Leu Leu Glu Ala Glu Gly Val Ser Val Thr
Val Ala Asp Ala Arg Phe 530 535 540 Ser Arg Pro Leu Asp Thr Gly Leu
Ile Asp Gln Leu Val Arg His His 545 550 555 560 Ala Ala Leu Val Thr
Val Glu Gln Gly Ala Met Gly Gly Phe Gly Ala 565 570 575 His Val Met
His Tyr Leu Ala Asn Ser Gly Gly Phe Asp Gly Gly Leu 580 585 590 Ala
Leu Arg Val Met Thr Leu Pro Asp Arg Phe Ile Glu Gln Ala Ser 595 600
605 Pro Glu Asp Met Tyr Ala Asp Ala Gly Leu Arg Ala Glu Asp Ile Ala
610 615 620 Ala Thr Ala Arg Gly Ala Leu Ala Arg Gly Arg Val Met Pro
Leu Arg 625 630 635 640 Gln Thr Ala Lys Pro Arg Ala Val 645 29 1944
DNA Rhodobacter sphaeroides CDS (1)..(1944) 29 atg acc aat ccc acc
ccg cga ccc gaa acc ccg ctt ttg gat cgc gtc 48 Met Thr Asn Pro Thr
Pro Arg Pro Glu Thr Pro Leu Leu Asp Arg Val 1 5 10 15 tgc tgc ccg
gcc gac atg aag gcg ctg agt gac gcc gaa ctg gag cgg 96 Cys Cys Pro
Ala Asp Met Lys Ala Leu Ser Asp Ala Glu Leu Glu Arg 20 25 30 ctg
gcc gac gaa gtg cgt tcc gag gtg att tcg gtc gtt gcc gag acg 144 Leu
Ala Asp Glu Val Arg Ser Glu Val Ile Ser Val Val Ala Glu Thr 35 40
45 gga gga cat ctg ggg tcc tcg ctg ggg gtg gtc gag ctg acc gtc gcg
192 Gly Gly His Leu Gly Ser Ser Leu Gly Val Val Glu Leu Thr Val Ala
50 55 60 ctg cat gca gtc ttc aac acg ccc acc gac aag ctc gtc tgg
gac gtg 240 Leu His Ala Val Phe Asn Thr Pro Thr Asp Lys Leu Val Trp
Asp Val 65 70 75 80 ggc cac cag tgc tac ccc cac aag atc ctc acc ggc
cgg cgc gag cag 288 Gly His Gln Cys Tyr Pro His Lys Ile Leu Thr Gly
Arg Arg Glu Gln 85 90 95 atg cgc acc ctg cgc cag aag ggc ggc ctc
tcg ggc ttc acc aag cgc 336 Met Arg Thr Leu Arg Gln Lys Gly Gly Leu
Ser Gly Phe Thr Lys Arg 100 105 110 tcg gaa tcc gcc tac gac ccg ttc
ggc gcg gcc cat tcc tcg acc tcg 384 Ser Glu Ser Ala Tyr Asp Pro Phe
Gly Ala Ala His Ser Ser Thr Ser 115 120 125 atc tcg gcc gcg ctc ggc
ttt gcc atg ggc cgc gag ctg ggc caa ccc 432 Ile Ser Ala Ala Leu Gly
Phe Ala Met Gly Arg Glu Leu Gly Gln Pro 130 135 140 gtg ggc gac acg
atc gcc gtg atc ggc gac ggc tcg atc acc gcg ggc 480 Val Gly Asp Thr
Ile Ala Val Ile Gly Asp Gly Ser Ile Thr Ala Gly 145 150 155 160 atg
gcc tac gag gcg ctg aac cac gcg ggc cat ctg aac aag cgc ctg 528 Met
Ala Tyr Glu Ala Leu Asn His Ala Gly His Leu Asn Lys Arg Leu 165 170
175 ttc gtg atc ctg
aac gac aat gac atg agc atc gcg ccg ccc gtg ggg 576 Phe Val Ile Leu
Asn Asp Asn Asp Met Ser Ile Ala Pro Pro Val Gly 180 185 190 gct ctg
gcg cgc tat ctc gtg aat ctc tcc tcg aag gcg ccc ttc gcc 624 Ala Leu
Ala Arg Tyr Leu Val Asn Leu Ser Ser Lys Ala Pro Phe Ala 195 200 205
acg ctg cgc gcg gcc gcc gac ggg ctc gag gcc tcg ctg ccg ggg ccg 672
Thr Leu Arg Ala Ala Ala Asp Gly Leu Glu Ala Ser Leu Pro Gly Pro 210
215 220 ctc cgc gac ggg gcg cgc cgg gcg cgc cag ctc gtg acc ggg atg
ccg 720 Leu Arg Asp Gly Ala Arg Arg Ala Arg Gln Leu Val Thr Gly Met
Pro 225 230 235 240 ggc ggg ggc acg ctc ttc gag gag ctg ggc ttc acc
tat gtg ggt ccc 768 Gly Gly Gly Thr Leu Phe Glu Glu Leu Gly Phe Thr
Tyr Val Gly Pro 245 250 255 atc gac ggc cac gac atg gag gcg ctg ctc
cag acg ctg cgc gcg gcg 816 Ile Asp Gly His Asp Met Glu Ala Leu Leu
Gln Thr Leu Arg Ala Ala 260 265 270 cgg gcc cgg acc acg ggg ccg gtg
ctc atc cat gtg gtc acg aag aag 864 Arg Ala Arg Thr Thr Gly Pro Val
Leu Ile His Val Val Thr Lys Lys 275 280 285 ggc aag ggc tac gcc cct
gcc gag aat gcc ccc gac aag tat cac ggg 912 Gly Lys Gly Tyr Ala Pro
Ala Glu Asn Ala Pro Asp Lys Tyr His Gly 290 295 300 gtg aac aag ttc
gac ccc gtc acg ggc gag cag aag aag tcg gtc gcc 960 Val Asn Lys Phe
Asp Pro Val Thr Gly Glu Gln Lys Lys Ser Val Ala 305 310 315 320 aac
gcg ccg aac tac acc aag gtc ttc ggc tcc acc ctg acc gag gag 1008
Asn Ala Pro Asn Tyr Thr Lys Val Phe Gly Ser Thr Leu Thr Glu Glu 325
330 335 gcc gcg cgc gat ccg cgc atc gtg gcc atc acc gcg gcc atg ccc
tcg 1056 Ala Ala Arg Asp Pro Arg Ile Val Ala Ile Thr Ala Ala Met
Pro Ser 340 345 350 ggc acc ggc gtc gac atc atg cag aag cgt ttc ccg
aac cgc gtc ttc 1104 Gly Thr Gly Val Asp Ile Met Gln Lys Arg Phe
Pro Asn Arg Val Phe 355 360 365 gac gtg ggc atc gcc gag cag cat gcc
gtg acc ttc gcg gcg ggc ctt 1152 Asp Val Gly Ile Ala Glu Gln His
Ala Val Thr Phe Ala Ala Gly Leu 370 375 380 gcc ggg gcc ggg atg aag
ccc ttc tgc gcg atc tat tcc tcg ttc ctg 1200 Ala Gly Ala Gly Met
Lys Pro Phe Cys Ala Ile Tyr Ser Ser Phe Leu 385 390 395 400 caa cgg
ggc tac gac cag atc gcc cat gac gtg gcg ctg cag aac ctt 1248 Gln
Arg Gly Tyr Asp Gln Ile Ala His Asp Val Ala Leu Gln Asn Leu 405 410
415 ccc gtc cgc ttc gtg atc gac cgg gcg ggg ctc gtg ggg gcc gac ggt
1296 Pro Val Arg Phe Val Ile Asp Arg Ala Gly Leu Val Gly Ala Asp
Gly 420 425 430 gcg acc cat gcg ggg gcc ttc gat gtg ggc ttc ctc acg
tcg ctg ccc 1344 Ala Thr His Ala Gly Ala Phe Asp Val Gly Phe Leu
Thr Ser Leu Pro 435 440 445 aat atg acc gtg atg gcc gcg gcc gac gag
gcc gag ctc atc cac atg 1392 Asn Met Thr Val Met Ala Ala Ala Asp
Glu Ala Glu Leu Ile His Met 450 455 460 atc gcc acc gcc gtg gcc ttc
gac gag ggc ccc att gcc ttc cgc ttc 1440 Ile Ala Thr Ala Val Ala
Phe Asp Glu Gly Pro Ile Ala Phe Arg Phe 465 470 475 480 ccg cgg ggc
gag ggg gtg ggc gtc gag atg ccc gag cgc ggg acc gtg 1488 Pro Arg
Gly Glu Gly Val Gly Val Glu Met Pro Glu Arg Gly Thr Val 485 490 495
ctg gaa ccc ggc cgg ggc cgc gtg gtg cgc gag ggg acg gat gtg gcg
1536 Leu Glu Pro Gly Arg Gly Arg Val Val Arg Glu Gly Thr Asp Val
Ala 500 505 510 atc ctt tcc ttc ggc gcg cat ctg cac gag gcc ttg cag
gcg gcg aaa 1584 Ile Leu Ser Phe Gly Ala His Leu His Glu Ala Leu
Gln Ala Ala Lys 515 520 525 ctc ctc gag gcc gag ggg gtg agc gtg acc
gtg gcc gac gcc cgc ttc 1632 Leu Leu Glu Ala Glu Gly Val Ser Val
Thr Val Ala Asp Ala Arg Phe 530 535 540 tcg cgc ccg ctc gac acg ggg
ctc att gac cag ctc gtg cgc cat cac 1680 Ser Arg Pro Leu Asp Thr
Gly Leu Ile Asp Gln Leu Val Arg His His 545 550 555 560 gcc gcg ctg
gtg acg gtg gag cag ggg gcc atg ggc ggc ttc ggc gct 1728 Ala Ala
Leu Val Thr Val Glu Gln Gly Ala Met Gly Gly Phe Gly Ala 565 570 575
cat gtc atg cac tat ctc gcc aat tcc ggc ggc ttc gac ggg ggc ctc
1776 His Val Met His Tyr Leu Ala Asn Ser Gly Gly Phe Asp Gly Gly
Leu 580 585 590 gcg ctc cgg gtc atg acg ctg ccc gac cgc ttc atc gag
cag gcg agc 1824 Ala Leu Arg Val Met Thr Leu Pro Asp Arg Phe Ile
Glu Gln Ala Ser 595 600 605 ccc gag gac atg tat gcc gat gcg ggg ctg
cgg gcc gag gat atc gcg 1872 Pro Glu Asp Met Tyr Ala Asp Ala Gly
Leu Arg Ala Glu Asp Ile Ala 610 615 620 gcc acc gcg cgg ggc gcg ctc
gcc cgg ggg cgc gtg atg ccg ctc cgg 1920 Ala Thr Ala Arg Gly Ala
Leu Ala Arg Gly Arg Val Met Pro Leu Arg 625 630 635 640 cag acg gca
aag ccg cgg gcg gtc 1944 Gln Thr Ala Lys Pro Arg Ala Val 645 30 394
PRT Rhodobacter sphaeroides 30 Met Arg Ser Leu Ser Ile Phe Gly Ala
Thr Gly Ser Ile Gly Glu Ser 1 5 10 15 Thr Phe Asp Leu Val Met Arg
Lys Gly Gly Pro Glu Ala Phe Arg Thr 20 25 30 Val Ala Leu Thr Gly
Gly Arg Asn Ile Arg Arg Leu Ala Glu Met Ala 35 40 45 Arg Ala Leu
Lys Ala Glu Leu Ala Val Thr Ala His Glu Asp Cys Leu 50 55 60 Pro
Ala Leu Arg Glu Ala Leu Ala Gly Thr Gly Thr Glu Val Ala Gly 65 70
75 80 Gly Ala Gln Ala Ile Ala Glu Ala Ala Asp Arg Pro Ala Asp Trp
Thr 85 90 95 Met Ser Ala Ile Val Gly Ala Ala Gly Leu Val Pro Gly
Met Arg Ala 100 105 110 Leu Lys His Gly Arg Thr Leu Ala Leu Ala Asn
Lys Glu Ser Leu Val 115 120 125 Thr Ala Gly Gln Leu Leu Met Arg Thr
Ala Gln Glu Asn Gly Ala Thr 130 135 140 Ile Leu Pro Val Asp Ser Glu
His Ser Ala Val Phe Gln Ala Leu Ala 145 150 155 160 Gly Glu Asp Thr
Ala Cys Val Glu Arg Val Ile Ile Thr Ala Ser Gly 165 170 175 Gly Pro
Phe Arg Asp Trp Ser Leu Glu Arg Ile Arg Ala Cys Thr Val 180 185 190
Ala Glu Ala Gln Ala His Pro Asn Trp Ser Met Gly Gln Arg Ile Ser 195
200 205 Ile Asp Ser Ala Ser Met Phe Asn Lys Ala Leu Glu Leu Ile Glu
Thr 210 215 220 Arg Glu Phe Phe Gly Phe Glu Pro Asp Arg Ile Glu Ala
Val Val His 225 230 235 240 Pro Gln Ser Ile Val His Ala Met Val Gly
Phe Cys Asp Gly Gly Leu 245 250 255 Met Ala His Leu Gly Pro Ala Asp
Met Arg His Ala Ile Gly Phe Ala 260 265 270 Leu Asn Trp Pro Gly Arg
Gly Glu Val Pro Val Ala Arg Ile Asp Leu 275 280 285 Ala Gln Ile Ala
Ser Leu Thr Phe Gln Lys Pro Asp Glu Glu Arg Phe 290 295 300 Pro Ala
Leu Arg Leu Ala Arg Asp Val Met Ala Ala Arg Gly Leu Ser 305 310 315
320 Gly Ala Ala Phe Asn Ala Ala Lys Glu Ile Ala Leu Asp His Phe Ile
325 330 335 Ala Gly Arg Ile Gly Phe Leu Asp Met Ala Ala Val Val Glu
Glu Thr 340 345 350 Leu Ala Gly Val Ser Thr Asp Pro Leu Phe Gly Lys
Val Pro Asp Ala 355 360 365 Leu Glu Glu Val Leu Ala Met Asp His Leu
Ala Arg Arg Ala Ala Glu 370 375 380 Glu Ala Ala Gly Leu Arg Gln Gln
Lys Arg 385 390 31 1182 DNA Rhodobacter sphaeroides CDS (1)..(1182)
31 atg cgc agc ctg tcg atc ttt ggg gcc acc ggc tcc atc ggc gaa tcc
48 Met Arg Ser Leu Ser Ile Phe Gly Ala Thr Gly Ser Ile Gly Glu Ser
1 5 10 15 acc ttc gac ctc gtc atg cgg aag ggc ggg ccc gag gcg ttc
cgc acc 96 Thr Phe Asp Leu Val Met Arg Lys Gly Gly Pro Glu Ala Phe
Arg Thr 20 25 30 gtc gct ctg acc ggc ggg cgc aac atc cgg cga ctg
gcc gaa atg gcg 144 Val Ala Leu Thr Gly Gly Arg Asn Ile Arg Arg Leu
Ala Glu Met Ala 35 40 45 cgt gcg ctg aag gcg gag ctt gcc gtc acc
gcg cat gag gac tgc ctg 192 Arg Ala Leu Lys Ala Glu Leu Ala Val Thr
Ala His Glu Asp Cys Leu 50 55 60 ccc gcg ctg cgc gag gcg ctg gcc
ggg acg ggc acc gag gtc gcg ggc 240 Pro Ala Leu Arg Glu Ala Leu Ala
Gly Thr Gly Thr Glu Val Ala Gly 65 70 75 80 ggg gcg cag gcc atc gcc
gag gcc gcc gac cgg ccg gcc gac tgg acc 288 Gly Ala Gln Ala Ile Ala
Glu Ala Ala Asp Arg Pro Ala Asp Trp Thr 85 90 95 atg tcg gcc atc
gtg ggc gcc gcg ggc ctc gtg ccc gga atg cgg gcg 336 Met Ser Ala Ile
Val Gly Ala Ala Gly Leu Val Pro Gly Met Arg Ala 100 105 110 ctg aag
cac ggc cgc acg ctg gcg ctc gcc aac aag gaa agc ctc gtg 384 Leu Lys
His Gly Arg Thr Leu Ala Leu Ala Asn Lys Glu Ser Leu Val 115 120 125
acg gca ggg caa ctc ctg atg cgg acg gcc cag gag aac ggc gcc acg 432
Thr Ala Gly Gln Leu Leu Met Arg Thr Ala Gln Glu Asn Gly Ala Thr 130
135 140 atc ctg ccg gtg gac agc gag cac tcc gcg gtc ttt cag gcg ctg
gcg 480 Ile Leu Pro Val Asp Ser Glu His Ser Ala Val Phe Gln Ala Leu
Ala 145 150 155 160 ggc gag gac acg gcc tgc gtc gag cgc gtc atc atc
acg gcg tcc ggc 528 Gly Glu Asp Thr Ala Cys Val Glu Arg Val Ile Ile
Thr Ala Ser Gly 165 170 175 ggg ccg ttc cgc gac tgg agc ctc gag cgc
atc cgc gcc tgc acc gtg 576 Gly Pro Phe Arg Asp Trp Ser Leu Glu Arg
Ile Arg Ala Cys Thr Val 180 185 190 gcc gag gcg cag gcc cat ccc aac
tgg tcc atg ggc cag cgg atc tcc 624 Ala Glu Ala Gln Ala His Pro Asn
Trp Ser Met Gly Gln Arg Ile Ser 195 200 205 atc gac agc gcc tcg atg
ttc aac aag gcg ctc gag ctg atc gag acg 672 Ile Asp Ser Ala Ser Met
Phe Asn Lys Ala Leu Glu Leu Ile Glu Thr 210 215 220 cgc gaa ttc ttc
ggc ttc gag ccg gac cgg atc gag gcg gtc gtc cat 720 Arg Glu Phe Phe
Gly Phe Glu Pro Asp Arg Ile Glu Ala Val Val His 225 230 235 240 ccg
caa tcc atc gtc cat gcg atg gtg ggc ttc tgc gac ggg ggc ctg 768 Pro
Gln Ser Ile Val His Ala Met Val Gly Phe Cys Asp Gly Gly Leu 245 250
255 atg gcc cat ctc ggc ccc gcc gac atg cgc cac gcc atc gga ttc gcg
816 Met Ala His Leu Gly Pro Ala Asp Met Arg His Ala Ile Gly Phe Ala
260 265 270 ctg aac tgg ccg ggt cgc ggc gag gtg ccc gtc gcc cgg atc
gac ctc 864 Leu Asn Trp Pro Gly Arg Gly Glu Val Pro Val Ala Arg Ile
Asp Leu 275 280 285 gca cag att gcg agc ctc acc ttc cag aag cct gac
gag gaa cgc ttt 912 Ala Gln Ile Ala Ser Leu Thr Phe Gln Lys Pro Asp
Glu Glu Arg Phe 290 295 300 ccg gcc ctg agg ctt gcg cga gac gtc atg
gcg gcg cgc ggc ctg tcg 960 Pro Ala Leu Arg Leu Ala Arg Asp Val Met
Ala Ala Arg Gly Leu Ser 305 310 315 320 ggc gcc gcc ttc aac gcg gcc
aag gag atc gcg ctc gat cat ttc atc 1008 Gly Ala Ala Phe Asn Ala
Ala Lys Glu Ile Ala Leu Asp His Phe Ile 325 330 335 gcc gga cgc atc
ggg ttt ctg gac atg gcg gcg gtg gtc gag gag acg 1056 Ala Gly Arg
Ile Gly Phe Leu Asp Met Ala Ala Val Val Glu Glu Thr 340 345 350 ctc
gcg ggc gtt tcg acc gac ccc ctg ttc gga aaa gtg ccc gac gcc 1104
Leu Ala Gly Val Ser Thr Asp Pro Leu Phe Gly Lys Val Pro Asp Ala 355
360 365 ctt gag gaa gtg ctg gcc atg gac cat ctc gct cgg aga gcg gca
gag 1152 Leu Glu Glu Val Leu Ala Met Asp His Leu Ala Arg Arg Ala
Ala Glu 370 375 380 gaa gcc gcc ggt ctc cgc cag cag aaa agg 1182
Glu Ala Ala Gly Leu Arg Gln Gln Lys Arg 385 390 32 23 DNA
Artificial Sequence Synthetic DNA 32 aagctgatct gggacgtggg gca 23
33 23 DNA Artificial Sequence Synthetic DNA 33 tgctatccgc
acaagatcct gac 23 34 23 DNA Artificial Sequence Synthetic DNA 34
gcatgctgtt ccgcgatgcc gac 23
* * * * *